Human-derived bacteria that induce proliferation or accumulation of regulatory T cells

ABSTRACT

Species of human-derived bacteria belonging to the Clostridia class have been shown to induce accumulation of regulatory T cells (Treg cells) in the colon and suppress immune functions. Pharmaceutical compositions containing these bacteria can be used to prevent and treat immune-mediated diseases such as autoimmune diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/362,097, filed May 30, 2014, which is a national stage filing under35 U.S.C. §371 of International Application PCT/JP2012/007687, filedNov. 29, 2012, which claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application No. 61/565,976, filed Dec. 1, 2011 and U.S.Provisional Application No. 61/607,360, filed Mar. 6, 2012. The entireteachings of the referenced applications are incorporated herein byreference.

TECHNICAL FIELD

The subject matter described herein relates to a composition ofhuman-derived bacteria that induces proliferation, accumulation, orproliferation and accumulation of regulatory T cells and whichcomprises, as an active component, (a) one or more (a, at least one)human-derived bacteria that belongs to the Clostridia class, (b) culturesupernatant of one or more (a, at least one) of the bacteria; (c) aphysiologically active substance derived from one or more of thebacteria or (d) a combination of any two or more of the foregoing. Italso relates to a method for inducing proliferation, accumulation orproliferation and accumulation of regulatory T cells. The composition,which comprises any of (a)-(d) above, is referred to as a bacterialcomposition. Moreover, the subject matter relates to a method fortreating or preventing at least one disease or condition that isresponsive to induction of regulatory T cells, such as autoimmunediseases, inflammatory diseases, and infectious diseases, byadministering the bacterial composition to an individual in needthereof.

BACKGROUND

Hundreds of species of commensal microorganisms are harbored in thegastrointestinal tracts of mammals, where they interact with the hostimmune system. Research using germ-free (GF) animals has shown that thecommensal microorganisms influence the development of the mucosal immunesystem, such as histogenesis of Peyer's patches (PPs) and isolatedlymphoid follicles (ILFs), secretion of antimicrobial peptides from theepithelium, and accumulation of unique lymphocytes in mucosal tissues,including immunoglobulin A-producing plasma cells, intraepitheliallymphocytes, IL-17-producing CD4-positive T cells (Th 17), andIL-22-producing NK-like cells (Non-Patent Literature (NPL) 1 to 7).Consequently, the presence of intestinal bacteria enhances protectivefunctions of the mucous membranes, enabling the host to mount robustimmune responses against pathogenic microbes invading the body. On theother hand, the mucosal immune system maintains unresponsiveness todietary antigens and harmless microbes (NPL Document 3). Abnormality inthe regulation of cross-talk between commensal bacteria and the immunesystem (intestinal dysbiosis) may lead to overly robust immune responseto environmental antigens and inflammatory bowel disease (IBD) mayresult (NPL 8 to 10).

Recent studies have shown that individual commensal bacteria controldifferentiation of their specific immune cells in the mucosal immunesystem. For example, Bacteroides fragilis, which is a commensalbacterium in humans, specifically induces a systemic Th1 cell responseand a mucosal IL-10-producing T cell response in mice, and plays a rolein protecting the host from colitis, which is caused by a pathogen (NPL3). Segmented filamentous bacteria, which are intestinal commensalbacteria in mice, induce mucosal Th17 cell response and enhanceresistance against infection of gastrointestinal tracts of the host witha pathogen (NPL 11 to 13). In addition, short-chain fatty acids derivedfrom several commensal bacteria are known to suppress intestinalinflammation (NPL 14). Moreover, it has been observed that the presenceof some species of intestinal microbiota greatly influences thedifferentiation of regulatory T cells (hereafter referred to as “Tregcells”) which help maintain homeostasis of the immune system. Althoughspecific species of murine bacterial commensals that can stronglystimulate Tregs have been identified (NPL 15), it is still unknownwhether species of human commensal bacteria exert an equivalentinfluence on the human immune system. Furthermore, the human intestinaltract harbors more than a thousand bacterial species, many of which havenot yet been cultured (NPL 16). It is not feasible to guess a prioriwhich ones, if any, might have an effect on Tregs.

In order to develop drugs, dietary supplements, or foods with beneficialimmune functions for human use, it is desirable to identify commensalmicroorganisms that naturally colonize humans and have immune-modulatingproperties. Furthermore, since many of the commensals in the humanmicrobiome have yet to be cultured, it is necessary to develop methodsto cultivate them so that they can be produced by traditional industrialfermentation processes and subsequently incorporated in pharmaceuticalor food formulations.

CD4⁺ T cells are regulatory T cells that have been identified as a cellsubset that suppresses immunity. A transcription factor, Foxp3, isexpressed in CD4⁺ T cells, which are known to play an important role inmaintaining immunological homeostasis (NPL 8, 9, 17, and 18).Foxp3-expressing cells are present in large numbers in the colon andonly Treg cells present locally in the colon constantly express IL-10,an immunosuppressive cytokine, at a high level (NPL 19). Animals havingCD4⁺ Foxp3⁺ cells from which IL-10 is specifically removed developinflammatory bowel disease (NPL 20).

Accordingly, there is a need to identify human-derived commensalbacterial species with the ability to strongly induce Treg cells toproduce IL-10 in the colon at a high level and to develop methods toculture such species. Such species could be used to enhanceimmunosuppression, which, in turn, can be applied to treatment ofautoimmune diseases, such as inflammatory bowel disease, inflammatorydiseases, allergies, or organ transplantation, among other diseases andconditions.

NON PATENT LITERATURE

-   [NPL 1] J. J. Cebra, “Am J Clin Nutr”, May, 1999, 69, 1046S-   [NPL 2] A. J. Macpherson, N. L. Harris, “Nat Rev Immunol”, Jun.    2004, 4, 478-   [NPL 3] J. L. Round, S. K. Mazmanian, “Nat Rev Immunol”, May 2009,    9, 313-   [NPL 4] D. Bouskra et al., “Nature”, Nov. 27, 2008, 456, 507-   [NPL 5] K. Atarashi et al., “Nature”, Oct. 9, 2008, 455, 808-   [NPL 6] Ivanov, I I et al., “Cell Host Microbe”, Oct. 16, 2008, 4,    337-   [NPL 7] S. L. Sanos et al., “Nat Immunol”, January 2009, 10, 83-   [NPL 8] M. A. Curotto de Lafaille, J. J. Lafaille, “Immunity”, May    2009, 30, 626-   [NPL 9] M. J. Barnes, F. Powrie, “Immunity”, Sep. 18, 2009, 31, 401-   [NPL 10] W. S. Garrett et al., “Cell”, Oct. 5, 2007, 131, 33-   [NPL 11] Ivanov, I I et al., “Cell”, Oct. 30, 2009, 139, 485.-   [NPL 12] V. Gaboriau-Routhiau et al., “Immunity”, Oct. 16, 2009, 31,    677-   [NPL 13] N. H. Salzman et al., “Nat Immunol”, 11, 76.-   [NPL 14] K. M. Maslowski et al., “Nature”, Oct. 29, 2009, 461, 1282-   [NPL 15] K. Atarashi et al., “Science”, Jan. 21, 2011, 331, 337-   [NPL 16] J. Quin et al., “Nature”, Mar. 4, 2010, 464, 59-   [NPL 17] L. F. Lu, A. Rudensky, “Genes Dev”, Jun. 1, 2009, 23, 1270-   [NPL 18] S. Sakaguchi, T. Yamaguchi, T. Nomura, M. Ono, “Cell”, May    30, 2008, 133, 775-   [NPL 19] C. L. Maynard et al., “Nat Immunol”, Sep. 2007, 8, 931-   [NPL 20] Y. P. Rubtsov et al., “Immunity”, April 2008, 28, 546

SUMMARY

The present compositions and methods have been made in view of theabove-described problems in the art. Described herein are methods ofidentifying and culturing intestinal commensal bacteria, isolated fromhumans, which induce, preferably strongly induce, the proliferation,accumulation, or proliferation and accumulation of regulatory T cells.Described are compositions, also referred to as bacterial compositions,that (1) comprise (a) one or more of the identified intestinal commensal(human-derived) bacteria; (b) a culture supernatant of one or more ofthe bacteria; (c) one or more physiologically active substance derivedfrom one or more of the bacteria or from one or more of the culturesupernatant; (d) or a combination of any two or three of (a)-(c) and (2)induce the proliferation and/or accumulation of regulatory T cells (Tregcells). Alternatively, a composition comprises (a) one or more of theidentified intestinal commensal (human-derived) bacteria; (b) a culturesupernatant of one or more of the bacteria; or (c) one or morephysiologically active substance derived from the bacteria or from theculture supernatant, wherein the composition induces proliferationand/or accumulation of regulatory T cells. In some embodiments, thecomposition comprises one or more of the identified intestinal commensal(human-derived) bacteria. In some embodiments, the composition comprisesa culture supernatant of one or more of the bacteria. In someembodiments, the composition comprises one or more physiologicallyactive substance derived from the bacteria or from the culturesupernatant. In some embodiments, the one or more bacteria or one ormore physiologically active substance derived from the bacteria is threeor more. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is five ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is seventeenor more. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria istwenty-three or more. In some embodiments, the one or more bacteria orone or more physiologically active substance derived from the bacteriais 23. In specific embodiments, the bacterial compositions induce, andpreferably strongly induce, proliferation, accumulation, orproliferation and accumulation of regulatory T cells that produce animmunosuppressive cytokine, such as IL-10, in the colon (e.g., the humancolon) at high levels. Such bacterial compositions are useful, forexample, to enhance immunosuppression and, as a result, to treatautoimmune diseases. Bacterial compositions comprise, as an activecomponent, at least one organism and/or at least one substance selectedfrom the group consisting of: Clostridium saccharogumia, Clostridiumramosum JCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662; a culturesupernatant of at least one (a, one or more) of the bacteriadescribed/listed herein; a physiologically active substance derived from(a, one or more) bacteria described/listed herein or any combination oftwo or three of the foregoing. Alternatively, bacterial compositionscomprise, as an active component, at least one organism or at least onesubstance selected from the group consisting of: Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662; a culturesupernatant of at least one (a, one or more) of the bacteriadescribed/listed herein; a physiologically active substance derived from(a, one or more) bacteria described/listed herein. In some embodiments,a bacterial composition comprises at least one organism selected fromthe group consisting of: Clostridium saccharogumia, Clostridium ramosumJCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662. In someembodiments, a bacterial composition comprises a culture supernatant ofat least one (a, one or more) of the bacteria described/listed herein.In some embodiments, a bacterial composition comprises a physiologicallyactive substance derived from (a, one or more) bacteria described/listedherein. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is three ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is five ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is 17 ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is 23 ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is 23.Bacterial compositions can comprise any bacteria (Clostridia or otherbacteria) that contain DNA comprising a nucleotide sequence havingsufficient homology with sequences provided herein and that exhibitsubstantially the same effect on regulatory T cells as that exerted byany one of the following: Clostridium saccharogumia, Clostridium ramosumJCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, and Anaerostipes caccae DSM 14662. In someembodiments, bacteria present in bacterial compositions have at least90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologywith sequences provided herein, such as, but not limited to, thenucleotide sequences designated OTU herein and listed, for example, atthe pages following the last Example. In specific embodiments, suchbacteria contain DNA comprising a nucleotide sequence that has at least90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologywith one or more DNA sequence designated herein as follows: OTU136;OTU46; OTU221; OTU9; OTU296; OTU21; OTU166; OTU73; OTU174; OTU14; OTU55;OTU337; OTU314; OTU195; OTU306; OTU87; OTU86; OTU152; OTU253; OTU259;OTU281; OTU288; OTU334; OTU359; OTU362; or OTU367. Alternatively,bacteria contain DNA comprising a nucleotide sequence that has at least90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologywith DNA of one or more of the following: Clostridium saccharogumia,Clostridium ramosum JCM1298, Clostridium ramosum, Flavonifractorplautii, Pseudoflavonifractor capillosus ATCC 29799, Clostridiumhathewayi, Clostridium saccharolyticum WM1, Bacteroides sp. MANG,Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, and Anaerostipes caccae DSM 14662. Inspecific embodiments, bacterial compositions comprise bacteria (such ashuman-derived bacteria) that contain DNA comprising a nucleotidesequence having at least 97%, 98% or 99% homology with sequencesprovided herein, such as, but not limited to, the nucleotide sequencesdesignated OTU herein and listed, for example, at the pages followingthe last Example. In specific embodiments, the bacteria in bacterialcompositions contain DNA comprising a nucleotide sequence that has atleast 97%, 98% or 99% homology with one or more DNA sequence designatedherein as follows: OTU136; OTU46; OTU221; OTU9; OTU296; OTU21; OTU166;OTU73; OTU174; OTU14; OTU55; OTU337; OTU314; OTU195; OTU306; OTU87;OTU86; OTU152; OTU253; OTU259; OTU281; OTU288; OTU334; OTU359; OTU362;or OTU367. Alternatively, the bacteria contain DNA comprising anucleotide sequence that has at least 97%, 98% or 99% homology with DNAof one or more of the following: Clostridium saccharogumia, Clostridiumramosum JCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662. Any of thebacteria of the Clostridia class can be present in spore form orvegetative form.

Solution of Problem

As described herein, among the more than a thousand species of bacteriain the human microbiome, there are several species that strongly inducethe accumulation of Tregs in the colon. As also described, although mostbacterial species present in fecal samples from healthy individuals donot have the ability to stimulate Tregs, species that belong to theClostridia class have the ability to cause a robust induction of Tregsin the colon. Moreover, the inventors have obtained in vitro cultures ofeach of the bacterial species identified and shown that inoculating micewith the in vitro cultured species also leads to a robust accumulationof Tregs in the colon.

As described herein, compositions that comprise, as an active component,(a) one or more of certain species of bacteria that belong to theClostridia class or bacteria that contain DNA comprising a nucleotidesequence having at least 90% homology with sequences provided herein, inspore form or in vegetative form; (b) a culture supernatant of one ormore such bacteria; (c) one or more physiologically active substancederived from (a) or (b); or (d) a combination of any two or three of(a), (b) and (c) and induce the proliferation and/or accumulation ofregulatory T cells (Treg cells) suppress immune functions.

More specifically:

One embodiment is a composition that induces proliferation, accumulationor both proliferation and accumulation of regulatory T cells, thecomposition comprising, as an active component, at least one organismand/or at least one substance selected from the group consisting of:Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662; a culture supernatant of at least one of the bacteriadescribed/listed herein, and a physiologically active substance derivedfrom a bacterium described/listed herein.

In some embodiments, the active component is one or more of Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662. In someembodiments, the active component is a culture supernatant of one ormore of the bacteria described/listed herein. In some embodiments, theactive component is one or more physiologically active substancesderived from a bacterium described/listed herein. In some embodiments,the one or more bacteria or one or more physiologically active substancederived from the bacteria is three or more. In some embodiments, the oneor more bacteria or one or more physiologically active substance derivedfrom the bacteria is five or more. In some embodiments, the one or morebacteria or one or more physiologically active substance derived fromthe bacteria is 17 or more. In some embodiments, the one or morebacteria or one or more physiologically active substance derived fromthe bacteria is 23 or more. In some embodiments, the one or morebacteria or one or more physiologically active substance derived fromthe bacteria is 23.

A bacterial composition as described herein comprises at least one ofthe following: one bacteria as described herein; at least one culturesupernatant obtained from culture in which one (or more) of the bacteriawas present (grown or maintained) or a fraction of such a supernatant;one or more physiologically active substance derived from one or morebacteria (such as from the bacteria named herein) or a combination ofany two or three of the foregoing. The term composition/bacterialcomposition refers to all such combinations.

The bacteria in the composition can be, for example, Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662 or anybacteria (such as human-derived bacteria) that contain DNA comprising atleast 90% homology (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% homology) with sequences provided herein, such as, but notlimited to, the nucleotide sequences designated OTU herein and listed,for example, at the pages following the last Example. In specificembodiments, the bacteria contain DNA comprising a nucleotide sequencethat has at least 97%, at least 98% or at least 99% homology with one ormore DNA sequence designated herein as follows: OTU136; OTU46; OTU221;OTU9; OTU296; OTU21; OTU166; OTU73; OTU174; OTU14; OTU55; OTU337;OTU314; OTU195; OTU306; OTU87; OTU86; OTU152; OTU253; OTU259; OTU281;OTU288; OTU334; OTU359; OTU362; or OTU367. Alternatively, the bacteriacontain DNA comprising a nucleotide sequence that has at least 97% (97%,98%, 99%, 100%) homology with DNA of one or more of the following:Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavornfractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, andAnaerostipes caccae DSM 14662.

In one embodiment, the composition induces regulatory T cells that aretranscription factor Foxp3-positive regulatory T cells orIL-10-producing regulatory T cells. In another embodiment, thecomposition has an immunosuppressive effect.

One embodiment is a pharmaceutical composition that inducesproliferation, accumulation or both proliferation and/or accumulation ofregulatory T cells and suppresses immune function. The pharmaceuticalcomposition comprises a bacterial composition described herein and apharmaceutically acceptable component, such as a carrier, a solvent or adiluent. In specific embodiments, such a pharmaceutical compositioncomprises (a) (1) one or more species of bacteria belonging to theClostridia class, as described herein, in spore form or in vegetativeform, (2) a culture supernatant of such bacteria, (3) a physiologicallyactive substance derived therefrom or (4) a combination of any two orthree of (1), (2) and (3) and (b) a pharmaceutically acceptablecomponent, such as carrier, a solvent or a diluent. In specificembodiments, (a) above is at least one organism or substance selectedfrom the group consisting of: Clostridium saccharogumia, Clostridiumramosum JCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662, a culturesupernatant of one or more of the bacteria, and a physiologically activesubstance derived from one or more of the bacteria. In some embodiments,(a) above is at least one organism selected from the group consistingof: Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662. In some embodiments, (1) above is a culturesupernatant of one or more of the bacteria. In some embodiments, (1)above is a physiologically active substance derived from one or more ofthe bacteria. In some embodiments, the at least one organism orsubstances is three or more. In some embodiments, the at least oneorganism or substances is five or more. In some embodiments, the atleast one organism or substances is 17 or more. In some embodiments, theat least one organism or substances is 23 or more. In some embodiments,the at least one organism or substances is 23. In further embodiments,(a)(1) above is bacteria (such as human-derived bacteria) that containDNA comprising at least 90% homology (e.g., 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% homology) with sequences providedherein, such as, but not limited to, the nucleotide sequences designatedOTU herein and listed, for example, at the pages following the lastExample. In specific embodiments of the pharmaceutical composition, thebacteria contain DNA comprising a nucleotide sequence that has at least97%, at least 98% or at least 99% homology with one or more DNA sequencedesignated herein as follows: OTU136; OTU46; OTU221; OTU9; OTU296;OTU21; OTU166; OTU73; OTU174; OTU14; OTU55; OTU337; OTU314; OTU195;OTU306; OTU87; OTU86; OTU152; OTU253; OTU259; OTU281; OTU288; OTU334;OTU359; OTU362; or OTU367. Alternatively, the bacteria in thepharmaceutical composition contain DNA comprising a nucleotide sequencethat has at least 97% (97%, 98%, 99%, 100%) homology with DNA of one ormore of the following: Clostridium saccharogumia, Clostridium ramosumJCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662.

The pharmaceutical composition induces the proliferation and/oraccumulation of regulatory T cells (Treg cells) and suppresses immunefunction.

Also provided is a method of inducing proliferation, accumulation orboth proliferation and accumulation of regulatory T cells in anindividual (e.g., an individual in need thereof, such as an individualin need of induction of proliferation and/or accumulation of regulatoryT cells). The method comprises administering to the individual abacterial composition described herein or a pharmaceutical compositioncomprising a bacterial composition described herein. In the method atleast one organism or substance selected from the group consisting of:Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662; a culture supernatant of one or more of the bacteriaor one or more component of the culture supernatant; a physiologicallyactive substance derived from one or more of the bacteria or acombination of two or three of the foregoing is administered to anindividual (also referred to as an individual in need thereof) who canbe a healthy individual or an individual in need of prevention,reduction or treatment of a condition or disease. For example, thecompositions described may be administered to an individual in need oftreatment, reduction in the severity of or prevention of a disease orcondition such as an autoimmune disease, an inflammatory disease, anallergic disease, and an infectious disease

Optionally, administration of the bacterial composition may be incombination with, or preceded by, a course of one or more antibiotics.

Optionally, administration of the bacterial composition may be incombination with administration of at least one prebiotic substance thatpreferentially favors the growth of the species in the bacterialcomposition over the growth of other human commensal bacterial species.In one embodiment, the prebiotic substance(s) is, for example, anondigestible oligosaccharide. In specific embodiments, the one or moreprebiotic substance(s) is selected from the group consisting of almondskin, inulin, oligofructose, raffinose, lactulose, pectin,hemicellulose, amylopectin, acetyl-Co A, biotin, beet molasses, yeastextracts, and resistant starch. Also contemplated herein is acomposition that comprises the bacterial composition and at least oneprebiotic substance.

The bacterial composition may be administered in combination with asubstance selected from the group consisting of corticosteroids,mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives,immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine,prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine,theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugsfor rhinitis, anti-cholinergic decongestants, mast-cell stabilizers,monoclonal anti-IgE antibodies, vaccines, anti-TNF inhibitors such asinfliximab, adalimumab, certolizumab pegol, golimumab, or etanercept,and combinations thereof. Also described herein is a composition thatcomprises the bacterial composition and at least one substance selectedfrom the group consisting of corticosteroids, mesalazine, mesalamine,sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs,cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate,antihistamines, glucocorticoids, epinephrine, theophylline, cromolynsodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis,anti-cholinergic decongestants, mast-cell stabilizers, monoclonalanti-IgE antibodies, vaccines, anti-TNF inhibitors such as infliximab,adalimumab, certolizumab pegol, golimumab, or etanercept, andcombinations thereof.

In a further embodiment, the bacterial composition can be used as anadjuvant to improve the efficacy of a vaccine formulation. For example,the bacterial composition can be used as an adjuvant to a vaccine forthe prophylaxis or treatment of an autoimmune disease or an allergicdisease. In some embodiments, a method for prophylaxis or treatment isprovided, the method comprising administering the bacterial compositionand adminstering a vaccine.

Assessment of the extent of induction of proliferation or accumulationof regulatory T cells that results from administration of a compositiondescribed herein can be carried out by a variety of approaches, such asby measurement of the number of Foxp3-expressing Tregs in a patientsample (such as a biopsy or a blood sample), promotion of IL-10expression, promotion of CTLA4 expression, promotion of IDO expression,suppression of IL-4 expression, or colonization of an individual withthe bacterial composition. The results of such assessments are used asan index of the induction of proliferation or accumulation of regulatoryT cells in the individual.

In one embodiment, administration of a composition described hereincauses induction of the regulatory T cells that are transcription factorFoxp3-positive regulatory T cells or IL-10-producing regulatory T cells.

The composition described herein can be administered by a variety ofroutes and in one embodiment, is administered orally to an individual inneed thereof, such as a patient in need thereof. The composition may beadministered in a number of oral forms, such as in spore-form (in a drypowder or dissolved in a liquid formulation), in enteric capsules, insachets, or in a food matrix, such as yogurt, or a drink.

Also provided is a method to predict a subject's response to treatment(predict whether the subject will or will not respond to treatment) withcompositions of the invention. The method comprises (a) obtaining a (atleast one, one or more) sample, such as a fecal sample or a colonicbiopsy, from a patient before he or she is treated with a bacterialcomposition described herein; (b) measuring or determining thepercentage or absolute counts in the sample of at least one bacterialspecies selected from the group consisting of: Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, and Anaerostipes caccae DSM 14662, therebyproducing a percentage or count, and (c) comparing the resultingpercentage or count (measurement) to a baseline value of the samemeasurement in a healthy subject, wherein a percentage or count in thesample obtained from the patient that is lower than the baseline valueindicates that the subject may respond favorably to administration ofthe bacterial composition. In some embodiments, the method furthercomprises (d) administering the bacterial composition to the patient ifthe percentage or count in the sample obtained from the patient is lowerthan the baseline value. Optionally, the method may further comprisemeasuring in a patient's sample (e.g., a fecal sample or a colonicbiopsy) the percentages or absolute counts of other commensal speciesthat belong to Clostridium Clusters IV and XIVa, but are not present inthe bacterial composition, wherein a value of the percentage or absolutecount (measurement) lower than baseline further indicates that thesubject may respond favorably to administration of the bacterialcompositions. In some embodiments, the method further comprisesadministering the bacterial composition to the patient if the value ofthe percentage or absolute count (measurement) is lower than baseline.In one embodiment, the patient being assessed suffers from inflammatorybowel disease or a C. difficile infection.

Also provided is a method of monitoring a subject's response totreatment with the bacterial compositions of the invention, comprising:(a) obtaining a (at least one) sample, such as a fecal sample or acolonic biopsy from a patient before treatment with a bacterialcomposition described herein; (b) obtaining, a (at least one)corresponding sample from the patient after treatment with a bacterialcomposition described herein; and (c) comparing the percentage orabsolute counts of at least one bacterial species selected from thegroup consisting of: Clostridium saccharogumia, Clostridium ramosumJCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662 in the sampleobtained in (a) with the percentage or absolute counts of the same atleast one bacterial species in the sample obtained in (b), wherein ahigher value in the sample obtained in (b) (after treatment with thebacterial composition) than in the sample obtained in (a) (beforetreatment) indicates that the subject has responded favorably totreatment (e.g. is a positive indicator of enhanced immunosuppression inthe subject). In some embodiments, the method further comprises (d)further administering the bacterial composition to the patient orceasing administration of the bacterial composition to the patient basedon the comparison in (c). Optionally, the method may further comprisemeasuring in the subject's samples the percentages or absolute counts ofother commensal species that belong to Clostridium Clusters IV and XIVa,but are not present in the bacterial composition, wherein a higher valueafter treatment than before treatment indicates that the subject hasresponded favorably to treatment.

Effects of Compositions and Methods Described Herein

The compositions described herein, which contain, as an activecomponent, selected bacteria belonging to the Clostridia class or otherbacteria, as described herein; a culture supernatant of such bacteria; aphysiologically active substance derived from such bacteria; or acombination of two or three of the foregoing are excellent at inducingthe proliferation or accumulation of regulatory T cells (Treg cells).

Immunity in an individual can be suppressed through administration ofthe subject composition, such as through ingestion of the bacterialcomposition in a food or beverage or as a dietary supplement or throughadministration of a pharmaceutical composition comprising the bacterialcomposition. The subject composition can be used, for example, toprevent or treat autoimmune diseases, allergic diseases, infectiousdiseases, as well as to suppress immunological rejection in organtransplantation or the like. In addition, if a food or beverage, such asa health food, comprises the subject composition, healthy individualscan ingest the composition easily and routinely. As a result, it ispossible to induce the proliferation and/or accumulation of regulatory Tcells and thereby to improve immune functions.

The composition described herein provides for a natural, long-lasting,patient-friendly, and benign treatment alternative for immune-mediatedconditions. For example, inflammatory bowel disease is currently managedwith synthetic drugs that may have severe side effects (such ascorticosteroids, TNF inhibitors), cannot be administered orally (such asTNF inhibitors), have inconvenient dosing involving several pills a day(such as mesalazine or sulfasalazine) or have limited efficacy andshort-lived effects (such as currently marketed probiotics, e.g.Lactobacillus GG, Lactobacillus acidophilus, Bifidobacterium longum,etc).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a histogram showing Foxp3 expression gated CD4 cells fromcolonic lamina propia (C LPL, left panel) and small intestinal laminapropia (SI LPL, right panel) of GF mice or GF mice colonized withuntreated (+huUT, n=4, numbering from #A1 to #A4) or chloroform-treated(+huChloro, n=4, numbering from #B1 to #B4) human feces. Numbers abovebracketed lines indicate the percentage of the population.

FIG. 1B is a histogram showing Helios expression in Foxp3⁺CD4⁺ cellsfrom colonic lamina propia (left panel) and small intestinal laminapropria (right panel) of GF mice or GF mice colonized with untreated(+huUT) or chloroform-treated (+huChloro) human feces. Numbers abovebracketed lines indicate the percentage of the population.

FIGS. 1C-1D are graphs showing, respectively, combined data for Foxp3expression in CD4+ cells, and for Helios expression in Foxp3⁺CD4⁺ cells,from colonic lamina propia (left panel) and small intestinal laminapropria (right panel) of GF mice or GF mice colonized with untreated(+huUT) or chloroform-treated (+huChloro) human feces. Each circle inFIG. 1C and FIG. 1D represents a separate animal, and error barsindicate the SD. *P<0.05; **P<0.001, unpaired t test.

FIG. 1E shows representative flow cytometry dot plots for theintracellular expressions of IL-17 and IFN− in CD4⁺ cells from coloniclamina propia (upper panel) and small intestinal lamina propria (lowerpanel) of GF mice or GF mice colonized with untreated (+huUT) orchloroform-treated (+huChloro) human feces. The number in each quadrantindicates the percentage of the population.

FIGS. 1F-1G show, respectively, combined data of all mice for IL-17 andIFN-expression in CD4+ cells from colonic lamina propia (left panel) andsmall intestinal lamina propria (right panel) of GF mice or GF micecolonized with untreated (+huUT) or chloroform-treated (+huChloro) humanfeces. Each circle in FIG. 1F and FIG. 1G represents a separate animal,and error bars indicate the SD. *P<0.05; ns, not significant (P>0.05),unpaired t test.

FIGS. 2A-2C shows representative plots (FIG. 2A) and combined data(FIGS. 2B-2C) for Foxp3 expression in CD4+ cells (upper panel in FIG.2A, left panel in FIG. 2B), or Helios expression in Foxp3+CD4+ cells(lower panel in FIG. 2A, right panel in FIG. 2C) for GF mice and GF miceorally inoculated (once a week for 4 weeks) with a suspension ofchloroform-treated human feces that had been previously autoclaved.Numbers above bracketed lines in FIG. 2A indicate the percentage of thepopulation. Each circle in FIG. 2B and FIG. 2C represents a separateanimal, and error bars indicate the SD. ns, not significant (P>0.05),unpaired t test.

FIGS. 3A-3B shows representative plots (FIG. 3A, data of mouse #C4 isshown here) and combined data (FIG. 3B) for Foxp3 expression in CD4+cells from colonic and small intestinal lamina propria lymphocytes forGF mice and GF mice orally inoculated with chloroform-treated humanfeces (+huChloro, n=7, numbering from #C1 to #C7). Numbers abovebracketed lines in FIG. 3A indicate the percentage of the population.Each circle in FIG. 3B represents a separate animal, and error barsindicate the SD. **P<0.001, unpaired t test.

FIGS. 4A-4B shows representative plots (FIG. 4A) and combined data (FIG.4B) for Foxp3 expression in CD4⁺ cells from colonic lamina propria (CLPL) and small intestinal lamina propria (SI LPL) for GF mice and GF(numbering from #D1 to #D6) that were co-housed with #C6 and #C7 ex-GFmice colonized with chloroform-treated human feces. Numbers abovebracketed lines in FIG. 4A indicate the percentage of the population.Each circle in FIG. 4B represents a separate animal, and error barsindicate the SD. **P<0.001, unpaired t test.

FIGS. 5A-5C shows representative plots and combined data for Foxp3expression in CD4+ cells (FIGS. 5A, 5B), or Helios expression inFoxp3⁺CD4⁺ cells (FIG. 5C) from colonic lamina propria (C LPL) and smallintestinal lamina propria (SI LPL) for GF mice, GF mice that wereinoculated with 2000-fold (+x2000, n=4, numbering from #E1 to #E4) or20000-fold (+x20000, n=8, numbering from #F1 to #F8) diluted fecalsuspension from #C4 mouse. Numbers above bracketed lines in FIG. 5Aindicate the percentage of the population. Each circle in FIG. 5B andFIG. 5C represents a separate animal, and error bars indicate the SD.*P<0.05; **P<0.001, unpaired t test.

FIGS. 6A-6D shows representative plots (FIGS. 6A, 6B) and combined data(FIGS. 6C, 6D) for Foxp3 expression in CD4⁺ cells (FIGS. 6A, 6C), orHelios expression in Foxp3⁺CD4⁺ cells (FIGS. 6B, 6D) from colonic laminapropria (C LPL) and small intestinal lamina propria (SI LPL) for GFmice, and GF mice that were inoculated with fecal suspension of #F3(n=5), #F7 (n=4) or #F8 (n=4) mouse. Numbers above bracketed lines inFIG. 6A and FIG. 6B indicate the percentage of the population. Eachcircle in FIG. 6C and FIG. 6D represents a separate animal, and errorbars indicate the SD. *P<0.05; **P<0.001, unpaired t test.

FIG. 7A-7C shows representative plots (FIG. 7A) and combined data (FIGS.7B, 7C) for Foxp3 expression in CD4+ cells (FIGS. 7A, 7B) or Heliosexpression in Foxp3⁺CD4⁺ cells for GF mice and GF mice that wereinoculated with 3 isolated strains of bacteria from cecal content of #F8mouse (n=4, numbering from #J1 to #J4). Numbers above bracketed lines inFIG. 7A indicate the percentage of the population. Each circle in FIG.7B and FIG. 7C represents a separate animal, and error bars indicate theSD. ns, not significant (P>0.05), unpaired t test.

FIG. 8 shows the relative abundances of OTUs having the same closestrelative in each cecal sample (bacterial DNA was extracted from thececal contents of mouse #A1, #C4, #F8, #G2, #H3, #I3, #J3 and #K3, shownin the bars). Total number of OTUs detected in each sample is depictedbelow the bar. The detected OTU names in sample #H3, #I3 or #K3, theirclosest relative and their similarity with the closest relative aredepicted in the right table.

FIGS. 9A-9C shows representative plots (FIG. 9A) and combined data(FIGS. 9B, 9C) for Foxp3 expression in CD4⁺ cells (FIGS. 9A, 9B), orHelios expression in Foxp3⁺CD4⁺ cells (FIGS. 9A, 9C) from colonic laminapropria (C LPL) and small intestinal lamina propria (SI LPL) for GF miceand GF mice that were inoculated with bacteria collections from cultureplate of cecal content of #G2 mouse (n=4, numbering from #K1 to #K4.Numbers above bracketed lines in FIG. 9A indicate the percentage of thepopulation. Each circle in FIG. 9B and FIG. 9C represents a separateanimal, and error bars indicate the SD. *P<0.05; **P<0.001, unpaired ttest.

FIGS. 10A-10C shows representative plots (FIG. 10A) and combined data(FIGS. 10B, 10C) for Foxp3 expression in CD4⁺ cells (FIG. 10A, 10B), orHelios expression in Foxp3⁺CD4⁺ cells (FIG. 10A, 10C) from coloniclamina propria (C LPL) and small intestinal lamina propria (SI LPL) forGF mice and GF mice that were inoculated with a mixture of 23 bacterialstrains that were isolated and shown in Table 2 (23 mix). Numbers abovebracketed lines in FIG. 10A indicate the percentage of the population.Each circle in FIG. 10B and FIG. 10C represents a separate animal, anderror bars indicate the SD. *P<0.05; **P<0.001, unpaired t test.

FIG. 11 shows a representative plot of the accumulation of Foxp3⁺CD4⁺cells in adult GF mice that were inoculated with 2×10⁴ to 2×10⁷-folddiluted caecal samples from +huChlo mice. Experiments were performedmore than twice. Error bars indicate SD. **P<0.01, *P<0.05, ascalculated by Student's t-test.

FIG. 12 shows a representative plot of the accumulation of Foxp3⁺CD4⁺cells in the colon of adult GF mice that were inoculated with a mixtureof 23 bacterial strains that were isolated and shown in Table 2(23-mix), chloroform-treated human feces (+huChlo) and Faecalibacteriumprausnitzii (+Faecali). Error bars indicate SD. **P<0.01, as calculatedby Student's t-test.

FIG. 13 shows a representative plot of the accumulation of Foxp3⁺CD4⁺cells in adult GF mice that were the secondary (+2×10⁴-re) and tertiary(+2×10⁴-re-re) recipients of inoculations with the caecal content of+2×10⁴ mice, and adult GF mice inoculated with 2×10⁴-fold diluted caecalsamples from +2×10⁴ mice (+(2×10⁴)²).

FIG. 14 shows the results of 16s rDNA pyrosequencing the caecal contentsfrom the defined mice (+hu, +huChlo, +2×10⁴, +2×10⁴-re, (+2×10⁴)²,+23-mix) using a 454 sequencer. The relative abundance of OTUs (%) inthe caecal bacterial community in each mouse and the closest strains inthe database and the corresponding isolated strain number for theindicated OTUs are shown.

FIG. 15 shows a representative plot of the accumulation of Foxp3⁺CD4⁺cells in the colons of adult IQI, BALB and B6 GF mice on inoculationwith a mixture of 17 bacterial strains that were isolated and shown inTable 4 (17-mix), **P<0.01, as calculated by Student's t-test.

FIG. 16 shows a representative plot of the accumulation of Foxp3⁺CD4⁺cells in adult IQI GF mice mono-colonized with each of the 17 strainslisted in Table 4 (17-mix).

FIG. 17 shows a representative plot of the accumulation of Foxp3⁺CD4⁺cells in adult IQI GF mice colonized with 3-mix, 5 mix-A, 5-mix-B,5-mix-C or 17-mix as listed in Table 4. Circles indicate individualanimals. Experiments wasere performed more than twice with similarresults. Error bars indicate SD. **P<0.01, *P<0.05, ns, not significant,as calculated by Student's t-test.

FIG. 18 shows a representative plot of the accumulation of Foxp3⁺CD4⁺cells in adult SPF mice repeatedly inoculated with 17-mix (SPF+17mix;n=5) or control (SPF+cont; n=6). **P<0.01, as calculated by Student'st-test.

FIG. 19 shows the effects of inoculation with 17-mix on an OVA model ofdiarrhea, as measured by a qualitative diarrhea score. *P<0.05, ascalculated by Student's t-test.

FIG. 20 shows the survival of adult mice inoculated with a mixture of 17bacterial strains listed in Table 4 (17-mix) following exposure totrinitrobenzene sulfonic acid (TNBS), an agent used in experimentalmodels of colitis.

FIG. 21 shows the relative abundance of each of the 17-mix strains inthe human fecal microbiota of ulcerative colitis and healthy subjects.The publically available reads of 15 healthy and 20 ulcerative colitissubjects in the MetaHIT database were aligned to the genome of the 17strains. The mean numbers of mapped reads in healthy and UC groups foreach of the 17 strain genomes are shown. Error bars represent SEM.*P<0.05, as calculated by the Student's t-test.

Table 1 shows the numbers of detected reads and the closest relativesfor each OTU obtained from classification of sequences (3400 reads foreach sample) resulting from 16srRNA coding gene amplification and PCRmetasequencing of bacterial DNA extracted from the cecal contents ofmouse #A1, #C4, #F8, #G2, #H3, #I3, #J3 and #K3 (classification on thebasis of sequence similarity, >97% identity to sequences in nucleic aciddatabases using BLAST)

Table 2 shows, for each of seventeen bacterial strains isolated from thececal contents of mouse #F8, #G2, #I1 and #K3 using BL agar or EG agarplates, the closest relative in known species, the maximum similaritywith the closest relative, its classification in the Clostridiaceaecluster, origin of mouse ID, and culture medium for isolation.

Table 3 shows, for each of 31 bacterial strains isolated from the caecalcontents of mouse #F8, #G2, #I1 and #K3 using BL agar or EG agar plates,the closest relative in known species, the maximum similarity with theclosest relative, the database used for BLAST search, and similaritybetween strains.

Table 4 shows 16S rDNA analysis for each of 31 strains that wereisolated. Bacterial DNA was isolated from each of the 31 strains and the16S rDNA of the isolates was amplified by colony-PCR. Each amplified DNAwas purified, sequenced, and aligned using the ClustalW softwareprogram. Based on the sequence of 16S rDNA for each strain, theirclosest species, % similarity with the closest species, and thesimilarity to other strains are shown. Strains that were included in the23-mix, 17-mix, 5-mixA, 5-mixB, 5-mixC, and 3-mix are marked in theright hand column.

DETAILED DESCRIPTION

<Composition Having Effect of Inducing Proliferation or Accumulation ofRegulatory T Cells>

Described herein is a composition that induces proliferation,accumulation of regulatory T cells or both proliferation andaccumulation of regulatory T cells. The composition comprises, as anactive ingredient, one or more of the following: a (at least one, one ormore) organism selected from the group consisting of: Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662, a culturesupernatant of one or more of the bacteria, a component of culturemedium in which a (at least one, one or more) bacterium described hereinhas grown, a physiologically active substance derived from a (at leastone; one or more) bacterium described herein; and a (at least one; oneor more) bacterium containing DNA comprising a nucleotide sequencehaving at least 97% homology to the nucleotide sequence of DNA of any ofthe bacterial species described herein, such as those listed above.Bacteria described herein were isolated from human fecal samples usingthe methods outlined in Examples 19 to 28.

The term “regulatory T cells” refers to T cells that suppress anabnormal or excessive immune response and play a role in immunetolerance. The regulatory T cells are typically transcription factorFoxp3-positive CD4-positive T cells. The regulatory T cells of thepresent invention also include transcription factor Foxp3-negativeregulatory T cells that are IL-10-producing CD4-positive T cells.

The term “induces proliferation or accumulation of regulatory T cells”refers to an effect of inducing the differentiation of immature T cellsinto regulatory T cells, which differentiation leads to theproliferation and/or the accumulation of regulatory T cells. Further,the meaning of “induces proliferation or accumulation of regulatory Tcells” includes in-vivo effects, in vitro effects, and ex vivo effects.All of the following effects are included: an effect of inducing in vivoproliferation or accumulation of regulatory T cells throughadministration or ingestion of the aforementioned bacteria belonging tothe Clostridia class, a culture supernatant of the bacteria orsupernatant component(s), or a physiologically active substance derivedfrom the bacteria; an effect of inducing proliferation or accumulationof cultured regulatory T cells by causing the aforementioned bacteriabelonging to the Clostridia class, a culture supernatant of the bacteriaor supernatant component(s), or a physiologically active substancederived from the bacteria to act on the cultured regulatory T cells; andan effect of inducing proliferation or accumulation of regulatory Tcells which are collected from a living organism and which are intendedto be subsequently introduced into a living organism, such as theorganism from which they were obtained or another organism, by causingthe aforementioned bacteria belonging to the Clostridia class, a culturesupernatant of the bacteria or supernatant component(s), or thephysiologically active substance derived from the bacteria to act on theregulatory T cells. The effect of inducing proliferation or accumulationof regulatory T cells can be evaluated, for example, as follows.Specifically, the aforementioned bacteria belonging to the Clostridiaclass, a culture supernatant of the bacteria or supernatantcomponent(s), or a physiologically active substance derived from thebacteria is orally administered to an experimental animal, such as agerm-free mouse, then CD4-positive cells in the colon are isolated, andthe ratio of regulatory T cells contained in the CD4-positive cells ismeasured by flow cytometry (refer to Example 7).

The regulatory T cells whose proliferation or accumulation is induced bythe composition of the present invention are preferably transcriptionfactor Foxp3-positive regulatory T cells or IL-10-producing regulatory Tcells.

In the present invention, “human-derived bacteria” means bacterialspecies that have been isolated from a fecal sample or from agastrointestinal biopsy obtained from a human individual or whoseancestors were isolated from a fecal sample or from a gastrointestinalbiopsy obtained from a human (e.g., are progeny of bacteria obtainedfrom a fecal sample or a gastrointestinal biopsy). For example, thebacterial species may have been previously isolated from a fecal sampleor from a gastrointestinal biopsy obtained from a human and cultured fora sufficient time to generate progeny. The progeny can then be furthercultured or frozen. The human-derived bacteria are naturally occurringcommensals that populate the gastrointestinal tract of humanindividuals, preferably healthy human individuals.

In the present invention, the term “Clostridia class” (as in“compositions containing bacteria belonging to the Clostridia class”)refers to a class of Gram+, obligate anaerobic bacteria belonging to theFirmicutes phylum that have the ability to form spores. It is importantto note that while currently most bacteria in this class are included inthe Clostridiales order, this categorization is still partly based onold methods and is likely to be redefined in the future based on newadvances in sequencing technologies that are enabling sequencing of thefull genomes of bacteria in this class. Table 2 provides a summary ofthe categorization of 17 abundant species belonging to the Clostridiaclass which have been identified by the inventors as strongTreg-inducers and cultured in vitro. All of these species fall, undercurrent categorization rules, in the Clostridiaceae family, and belongto clusters IV, XIVa, XVI, and XVIII.

The composition of the present invention may include one strain alone(only one strain) of any of the aforementioned bacterial species, buttwo or more strains of the bacteria can be used together. For example,one, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen or seventeen of the strainslisted in Table 2 or Table 4, in any combination, can be used togetherto affect regulatory T cells. In some embodiments, the 23, 17, 5, or 3species mixes listed in Table 4 can be used together (and administeredin one or several compositions) to affect regulatory T cells. In someembodiments, the following strains can be combined (the compositioncomprises): strain 1 (OTU136, closest species: Clostridiumsaccharogumia, Clostridium ramosum JCM1298), strain 3 (OTU221, closestspecies: Flavonifractor plautii, Pseudoflavonifractor capillosus ATTC29799), strain 4 (OTU9, closest species: Clostridium hathewayi,Clostridium saccharolyticum WM1), strain 5 (OTU296, closest species:Clostridium scindens, Lachnospiraceae bacterium 5_1_57FAA), strain 6(OTU21, closest species: Blautia coccoides, Lachnospiraceae bacterium6_1_63FAA), strain 7 (OUT 166, closest species: Clostridium sp.,Clostridium bolteae ATCC BAA-613), strain 8 (OTU73, closest species: cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A), strain 9(OTU174, closest species: Clostridium indolis, Anaerostipes caccae DSM14662), strain 10 (OTU166, closest species: Clostridium bolteae,Clostridiu bolteae ATCC BAA-613), strain 12 (OTU55, closest species:Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1), strain 13 (OTU337, closest species: Anaerotruncuscolihominis, Anaerotruncus colihominis DSM 17241), strain 14 (OTU314,closest species: Ruminococcus sp. ID8, Lachnospiraceae bacterium2_1_46FAA), strain 15 (OTU195, closest species: Clostridium lavalense,Clostridium asparagiforme DSM 15981), strain 16 (OTU306, closestspecies: Clostridium symbiosum, Clostridium symbiosum WAL-14163), strain18 (OTU46, closest species: Clostridium ramosum, Clostridium ramosum),strain 21 (OTU87, closest species: Eubacterium contortum, Clostridiumsp. D5), strain 23 (OTU152, closest species: Lachnospiraceae bacteriumDJF_VP30, Lachnospiraceae bacterium 3_1_57FAA_CT1), strain 24 (OTU253,closest species: Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes), strain 25 (OTU259, closest species: Eubacteriumcontortum, Clostridium sp. D5), strain 26 (OTU281, closest species:Clostridium scindens, Lachnospiraceae bacterium 5_1_57FAA), strain 27(OTU288, closest species: Lachnospiraceae bacterium A4, Lachnospiraceaebacterium 3_1_57FAA_CT1), strain 28 (OTU344, closest species:Clostridium sp. 316002/08, Clostridiales bacterium 1_7_47FAA), andstrain 29 (OTU359, closest species: Lachnospiraceae bacterium A4,Lachnospiraceae bacterium 3_1_57FAA_CT1) as described in Table 4.

In some embodiments, the following strains can be combined (thecomposition comprises: strain 1 (OTU136, closest species: Clostridiumsaccharogumia, Clostridium ramosum JCM1298), strain 3 (OTU221, closestspecies: Flavonifractor plautii, Pseudoflavonifractor capillosus ATTC29799), strain 4 (OTU9, closest species: Clostridium hathewayi,Clostridium saccharolyticum WM1), strain 6 (OTU21, closest species:Blautia coccoides, Lachnospiraceae bacterium 6_1_63FAA), strain 7 (OUT166, closest species: Clostridium sp., Clostridium bolteae ATCCBAA-613), strain 8 (OTU73, closest species: cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A), strain 9 (OTU174, closestspecies: Clostridium indolis, Anaerostipes caccae DSM 14662), strain 13(OTU337, closest species: Anaerotruncus colihominis, Anaerotruncuscolihominis DSM 17241), strain 14 (OTU314, closest species: Ruminococcussp. ID8, Lachnospiraceae bacterium 2_1_46FAA), strain 15 (OTU195,closest species: Clostridium lavalense, Clostridium asparagiforme DSM15981), strain 16 (OTU306, closest species: Clostridium symbiosum,Clostridium symbiosum WAL-14163), strain 18 (OTU46, closest species:Clostridium ramosum, Clostridium ramosum), strain 21 (OTU87, closestspecies: Eubacterium contortum, Clostridium sp. D5), strain 26 (OTU281,closest species: Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA), strain 27 (OTU288, closest species: Lachnospiraceaebacterium A4, Lachnospiraceae bacterium 3_1_57FAA_CT1), strain 28(OTU344, closest species: Clostridium sp. 316002/08, Clostridialesbacterium 1_7_47FAA), and strain 29 (OTU359, closest species:Lachnospiraceae bacterium A4, Lachnospiraceae bacterium 3_1_57FAA_CT1)as described in Table 4.

In some embodiments, the following strains can be combined (thecomposition comprises): strain 1 (OTU136, closest species: Clostridiumsaccharogumia, Clostridium ramosum JCM1298), strain 4 (OTU9, closestspecies: Clostridium hathewayi, Clostridium saccharolyticum WM1), strain16 (OTU306, closest species: Clostridium symbiosum, Clostridiumsymbiosum WAL-14163), strain 27 (OTU288, closest species:Lachnospiraceae bacterium A4, Lachnospiraceae bacterium 3_1_57FAA_CT1),and strain 29 (OTU359, closest species: Lachnospiraceae bacterium A4,Lachnospiraceae bacterium 3_1_57FAA_CT1) as described in Table 4. Insome embodiments, the following strains can be combined: strain 6(OTU21, closest species: Blautia coccoides, Lachnospiraceae bacterium6_1_63FAA), strain 8 (OTU73, closest species: cf. Clostridium sp.MLG055, Erysipelotrichaceae bacterium 2_2_44A), strain 13 (OTU337,closest species: Anaerotruncus colihominis, Anaerotruncus colihominisDSM 17241), strain 14 (OTU314, closest species: Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA), and strain 26 (OTU281, closestspecies: Clostridium scindens, Lachnospiraceae bacterium 5_1_57FAA) asdescribed in Table 4. In some embodiments, the following strains can becombined: strain 3 (OTU221, closest species: Flavonifractor plautii,Pseudoflavonifractor capillosus ATTC 29799), strain 7 (OUT 166, closestspecies: Clostridium sp., Clostridium bolteae ATCC BAA-613), strain 9(OTU174, closest species: Clostridium indolis, Anaerostipes caccae DSM14662), strain 15 (OTU195, closest species: Clostridium lavalense,Clostridium asparagiforme DSM 15981), and strain 28 (OTU344, closestspecies: Clostridium sp. 316002/08, Clostridiales bacterium 1_7_47FAA)as described in Table 4 In some embodiments, the following strains canbe combined: strain 1 (OTU136, closest species: Clostridiumsaccharogumia, Clostridium ramosum JCM1298), strain 2 (OTU46, closestspecies: Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799) and strain 3 (OTU221, closest species: Flavonifractor plautii,Pseudoflavonifractor capillosus ATTC 29799) as described in Table 4. Theuse of multiple strains of the aforementioned species of bacteria,preferably belonging to the Clostridium cluster XIVa or the cluster IVin combination can bring about an excellent effect on regulatory Tcells. In addition to the bacteria belonging to clusters XIVa and IV,Clostridium ramosum, Clostridium saccharogumia (belonging to clusterXVIII) and cf. Clostridium sp. MLG055 (belonging to cluster XVI) canalso be used. If more than one strain of bacteria is used (e.g., one ormore strain belonging to cluster XIVa, one or more strain belonging tocluster IV, one or more strain belonging to clusters XVIII or XVI or acombination of any of the foregoing), the number and ratio of strainsused can vary widely. The number and ratio to be used can be determinedbased on a variety of factors (e.g., the desired effect, such asinduction or inhibition of proliferation or accumulation of regulatory Tcells; the disease or condition to be treated, prevented or reduced inseverity; the age or gender of the recipient; the typical amounts of thestrains in healthy humans). The strains can be present in a singlecomposition, in which case they can be consumed or ingested together (ina single composition), or can be present in more than one composition(e.g., each can be in a separate composition), in which case they can beconsumed individually or the compositions can be combined and theresulting combination (combined compositions) consumed or ingested. Anynumber or combination of the strains that proves effective (e.g., anynumber from one to 22, such as 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to3, 1 to 2, and any number therebetween or one to 23, such as 1 to 23, 3to 23, 5 to 23, 1 to 20, 1 to 17, 3 to 17, 5 to 17, 1 to 15, 1 to 10, 1to 5, 3 to 5, 1 to 3, 1 to 2, and any number therebetween) can beadministered. In certain embodiments of the present invention, acombination of some or all of the 22 or 23 (e.g., the 23 strains inExample 32 and Table 4) strains described in the present disclosure isused. For example, at least one, two or more, three, three or more,four, four or more, five, five or more, six, six or more or any othernumber of the 22 or 23 described strains, including 22 or 23 strains,can be used. In some embodiments, the specific combinations of 3, 5, 17,or 23 strains described in Table 4 can be used (the compositioncomprises combinations of 3, 5, 17 or 23 strains described in Table 4).They can be used in combination with one another and in combination withstrains not described in the cited reference.

Cells of bacteria belonging to the Clostridia class, such as thesespecifically described herein, can be used in spore form or invegetative form. From the viewpoint of stability to high temperature andpressure conditions, extended shelf life, ease of handling, resistanceto antibiotics, and lack of need for a cold chain storage anddistribution, the bacteria may be preferably in the form of spore. Fromthe viewpoint of abiding by the directives of certain manufacturingorganizations that do not tolerate spore contamination in theirfacilities, the bacteria may alternatively be produced (and lateradministered) in the form of vegetative cells.

The term the “physiologically active substance derived from bacteriabelonging to the Clostridia class” of the present invention includessubstances contained in the bacteria, secretion products of thebacteria, and metabolites of the bacteria. Such a physiologically activesubstance can be identified by purifying an active component from thebacteria, a culture supernatant thereof, or intestinal tract contents inthe intestinal tract of a mouse in which only bacteria belonging to theClostridia class are colonized by an already known purification method.

“Chloroform treatment” of a fecal sample obtained from a human is amethod that isolates the bacteria in the fecal sample that have theability to form spores, and is not particularly limited, as long as thespore-forming fraction is obtained by treating feces of a human withchloroform (for example, 3% chloroform), and has the effect of inducingproliferation or accumulation of regulatory T cells, including mammalianregulatory T cells such as murine regulatory T cells and humanregulatory T cells.

When the aforementioned “bacteria belonging to the Clostridia class” arecultured in a medium, substances contained in the bacteria, secretionproducts and metabolites produced by the bacteria are released from thebacteria. The meaning of the active ingredient “culture supernatant ofthe bacteria” in the composition of the present invention includes suchsubstances, secretion products, and metabolites. The culture supernatantis not particularly limited, as long as the culture supernatant has theeffect of inducing proliferation or accumulation of regulatory T cells.Examples of the culture supernatant include a protein fraction of theculture supernatant, a polysaccharide fraction of the culturesupernatant, a lipid fraction of the culture supernatant, and alow-molecular weight metabolite fraction of the culture supernatant.

The bacterial composition may be administered in the form of apharmaceutical composition, a dietary supplement, or a food or beverage(which may also be an animal feed), or may be used as a reagent for ananimal model experiment. The pharmaceutical composition, the dietarysupplement, the food or beverage, and the reagent induce proliferationor accumulation of regulatory T cells. An example presented hereinrevealed that regulatory T cells (Treg cells) induced by bacteria or thelike belonging to the Clostridia class suppressed the proliferation ofeffector T-cells. The composition of the present invention can be usedsuitably as a composition having an immunosuppressive effect. Theimmunosuppressive effect can be evaluated, for example, as follows.Regulatory T cells isolated from an experimental animal, such as amouse, to which the composition of the present invention is orallyadministered are caused to act on effector T-cells (CD4⁺ CD25⁻ cells)isolated from the spleen, and the proliferation ability thereof ismeasured by using the intake amount of [³H]-thymidine as an index (referto Example 14).

The bacterial composition of the present invention can be used, forexample, as a pharmaceutical composition for preventing or treating(reducing, partially or completely, the adverse effects of) anautoimmune disease, such as chronic inflammatory bowel disease, systemiclupus erythematosus, rheumatoid arthritis, multiple sclerosis, orHashimoto's disease; an allergic disease, such as a food allergy,pollenosis, or asthma; an infectious disease, such as an infection withClostridium difficile; an inflammatory disease such as a TNF-mediatedinflammatory disease (e.g., an inflammatory disease of thegastrointestinal tract, such as pouchitis, a cardiovascular inflammatorycondition, such as atherosclerosis, or an inflammatory lung disease,such as chronic obstructive pulmonary disease); a pharmaceuticalcomposition for suppressing rejection in organ transplantation or othersituations in which tissue rejection might occur; a supplement, food, orbeverage for improving immune functions; or a reagent for suppressingthe proliferation or function of effector T-cells.

More specific examples of target diseases for which the composition isuseful for treatment (reducing adverse effects or prevention) includeautoimmune diseases, allergic diseases, infectious diseases, andrejection in organ transplantations, such as inflammatory bowel disease(IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis,rheumatoid arthritis, Type I diabetes, multiple sclerosis, graft vs.host disease following bone marrow transplantation, osteoarthritis,juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis,reactive arthritis, spondy loarthropathy, systemic lupus erythematosus,insulin dependent diabetes mellitus, thyroiditis, asthma, psoriasis,dermatitis scleroderma, atopic dermatitis, graft versus host disease,acute or chronic immune disease associated with organ transplantation,sarcoidosis, atherosclerosis, disseminated intravascular coagulation,Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatiguesyndrome, Wegener's granulomatosis, Henoch-Schoenlejn purpurea,microscopic vasculitis of the kidneys, chronic active hepatitis,uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, polyglandular deficiencytype I syndrome and polyglandular deficiency type II syndrome, Schmidt'ssyndrome, adult (acute) respiratory distress syndrome, alopecia,alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease,psoriatic arthropathy, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarhopathy, atheromatous disease/arteriosclerosis,allergic colitis, atopic allergy, food allergies such as peanut allergy,tree nut allergy, egg allergy, milk allergy, soy allergy, wheat allergy,seafood allergy, shellfish allergy, or sesame seed allergy, autoimmunebullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,linear IgA disease, autoimmune haemolytic anaemia, Coombs positivehaemolytic anaemia, acquired pernicious anaemia, juvenile perniciousanaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneouscandidiasis, giant cell arteritis, primary sclerosing hepatitis,cryptogenic autoimmune hepatitis, Acquired Immunodeficiency DiseaseSyndrome, Acquired Immunodeficiency Related Diseases, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, fibrotic lung disease, cryptogenic fibrosingalveolitis, postinflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjogren's disease associated lung disease,ankylosing spondy litis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, radiation fibrosis, bronchiolitis obliterans,chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease,postinfectious interstitial lung disease, gouty arthritis, autoimmunehepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoidhepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis),autoimmune mediated hypoglycemia, type B insulin resistance withacanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, idiopathic leucopenia, autoimmune neutropenia, renaldisease NOS, glomerulonephritides, microscopic vasulitis of the kidneys,discoid lupus, erythematosus, male infertility idiopathic or NOS, spermautoimmunity, multiple sclerosis (all subtypes), insulindependentdiabetes mellitus, sympathetic ophthalmia, pulmonary hypertensionsecondary to connective tissue disease, Goodpasture's syndrome,pulmonary manifestation of polyarteritis nodosa, acute rheumatio fever,rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu'sdisease/arteritis, autoimmune thrombocytopenia, idiopathicthrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrousautoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmunehypothyroidism, primary myxoedema, phacogenic uveitis, primaryvasculitis, vitiligo, allergic rhinitis (pollen allergies), anaphylaxis,pet allergies, latex allergies, drug allergies, allergicrhinoconjuctivitis, eosinophilic esophagitis, hypereosinophilicsyndrome, eosinophilic gastroenteritis cutaneous lupus erythematosus,eosinophilic esophagitis, hypereosinophilic syndrome, and eosinophilicgastroenteritis, and diarrhea.

Additional examples of target diseases for which the composition isuseful for treatment include colon cancer, cystic fibrosis, celiacdisease, Type 2 diabetes, and autism-related immunopathologies. Thesediseases are characterized by a reduction of Clostridium Clusters IV andXIV in the gastrointestinal microbiota.

Compositions described herein can also be used as a pharmaceuticalcomposition for preventing or treating infectious diseases in anindividual whose resistance to the infectious diseases is impaired, forexample because of damage due to excessive inflammation caused by theimmunity or due to an alteration of the patient's microbiome. Examplesof infectious pathogens that impair maintenance or recovery ofhomeostasis of a host, and which eventually bring about suchimmunopathological tissue damage include Salmonella, Shigella,Clostridium difficile, Mycobacterium (which cause the diseasetuberculosis), protozoa (which cause malaria), filarial nematodes (whichcause the disease filariasis), Schistosoma (which causeschistosomiasis), Toxoplasma (which cause the disease toxoplasmosis),Leishmania (which cause the disease leishmaniasis), HCV and HBV (whichcause the disease hepatitis C and hepatitis B), and herpes simplexviruses (which cause the disease herpes).

Pharmaceutical preparations can be formulated from the bacterialcompositions described by drug formulation methods known to those ofskill in the art. For example, the composition can be used orally orparenterally in the form of capsules, tablets, pills, sachets, liquids,powders, granules, fine granules, film-coated preparations, pellets,troches, sublingual preparations, chewables, buccal preparations,pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments,plasters, cataplasms, transdermal absorption systems, lotions,inhalations, aerosols, injections, suppositories, and the like.

For formulating these preparations, the bacterial compositions can beused in appropriate combination with carriers that are pharmacologicallyacceptable or acceptable for ingestion, such as in a food or beverage,including one or more of the following: sterile water, physiologicalsaline, vegetable oil, solvent, a base material, an emulsifier, asuspending agent, a surfactant, a stabilizer, a flavoring agent, anaromatic, an excipient, a vehicle, a preservative, a binder, a diluent,a tonicity adjusting agent, a soothing agent, a bulking agent, adisintegrating agent, a buffer agent, a coating agent, a lubricant, acolorant, a sweetener, a thickening agent, a flavor corrigent, asolubilizer, and other additives.

A pharmaceutical preparation or formulation and particularly apharmaceutical preparation for oral administration, comprises anadditional component that enables efficient delivery of the bacterialcomposition of the present invention to the colon, in order to moreefficiently induce proliferation or accumulation of regulatory T cellsin the colon. A variety of pharmaceutical preparations that enable thedelivery of the bacterial composition to the colon can be used. Examplesthereof include pH sensitive compositions, more specifically, bufferedsachet formulations or enteric polymers that release their contents whenthe pH becomes alkaline after the enteric polymers pass through thestomach. When a pH sensitive composition is used for formulating thepharmaceutical preparation, the pH sensitive composition is preferably apolymer whose pH threshold of the decomposition of the composition isbetween about 6.8 and about 7.5. Such a numeric value range is a rangein which the pH shifts toward the alkaline side at a distal portion ofthe stomach, and hence is a suitable range for use in the delivery tothe colon.

Another embodiment of a pharmaceutical preparation useful for deliveryof the bacterial composition to the colon is one that ensures thedelivery to the colon by delaying the release of the contents (e.g., thebacterial composition) by approximately 3 to 5 hours, which correspondsto the small intestinal transit time. In one embodiment of apharmaceutical preparation for delayed release, a hydrogel is used as ashell. The hydrogel is hydrated and swells upon contact withgastrointestinal fluid, with the result that the contents areeffectively released (released predominantly in the colon). Delayedrelease dosage units include drug-containing compositions having amaterial which coats or selectively coats a drug or active ingredient tobe administered. Examples of such a selective coating material includein vivo degradable polymers, gradually hydrolyzable polymers, graduallywater-soluble polymers, and/or enzyme degradable polymers. A widevariety of coating materials for efficiently delaying the release isavailable and includes, for example, cellulose-based polymers such ashydroxypropyl cellulose, acrylic acid polymers and copolymers such asmethacrylic acid polymers and copolymers, and vinyl polymers andcopolymers such as polyvinylpyrrolidone.

Examples of the composition enabling the delivery to the colon furtherinclude bioadhesive compositions which specifically adhere to thecolonic mucosal membrane (for example, a polymer described in thespecification of U.S. Pat. No. 6,368,586) and compositions into which aprotease inhibitor is incorporated for protecting particularly abiopharmaceutical preparation in the gastrointestinal tracts fromdecomposition due to an activity of a protease.

An example of a system enabling the delivery to the colon is a system ofdelivering a composition to the colon by pressure change in such a waythat the contents are released by utilizing pressure change caused bygeneration of gas in bacterial fermentation at a distal portion of thestomach. Such a system is not particularly limited, and a more specificexample thereof is a capsule which has contents dispersed in asuppository base and which is coated with a hydrophobic polymer (forexample, ethyl cellulose).

Another example of the system enabling the delivery to the colon is asystem of delivering a composition to the colon, the system beingspecifically decomposed by an enzyme (for example, a carbohydratehydrolase or a carbohydrate reductase) present in the colon. Such asystem is not particularly limited, and more specific examples thereofinclude systems which use food components such as non-starchpolysaccharides, amylose, xanthan gum, and azopolymers.

When used as a pharmaceutical preparation, the bacterial composition maybe used in combination with an already known pharmaceutical compositionfor use in immunosuppression. In some embodiments, the pharmaceuticalpreparation can comprise both the bacterial composition and the alreadyknown pharmaceutical composition. Such a known pharmaceuticalcomposition is not particularly limited, and may be at least onetherapeutic composition selected from the group consisting ofcorticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazinederivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine,azathiopurine, prednisone, methotrexate, antihistamines,glucocorticoids, epinephrine, theophylline, cromolyn sodium,anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergicdecongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies,vaccines (preferably vaccines used for vaccination where the amount ofan allergen is gradually increased), anti-TNF inhibitors such asinfliximab, adalimumab, certolizumab pegol, golimumab, or etanercept,and combinations thereof. It is preferable to use these therapeuticcompositions in combination with the bacterial composition describedherein. The bacterial composition can also be used as an adjuvant toimprove the efficacy of a vaccine formulation such as a vaccine for theprophylaxis or treatment of an autoimmune disease or an allergicdisease.

The bacterial composition can be used as a food or beverage, such as ahealth food or beverage, a food or beverage for infants, a food orbeverage for pregnant women, athletes, senior citizens or otherspecified group, a functional food, a beverage, a food or beverage forspecified health use, a dietary supplement, a food or beverage forpatients, or an animal feed. Specific examples of the foods andbeverages include various beverages such as juices, refreshingbeverages, tea beverages, drink preparations, jelly beverages, andfunctional beverages; alcoholic beverages such as beers;carbohydrate-containing foods such as rice food products, noodles,breads, and pastas; paste products such as fish hams, sausages, pasteproducts of seafood; retort pouch products such as curries, food dressedwith a thick starchy sauces, and Chinese soups; soups; dairy productssuch as milk, dairy beverages, ice creams, cheeses, and yogurts;fermented products such as fermented soybean pastes, yogurts, fermentedbeverages, and pickles; bean products; various confectionery productssuch as Western confectionery products including biscuits, cookies, andthe like, Japanese confectionery products including steamed bean-jambuns, soft adzuki-bean jellies, and the like, candies, chewing gums,gummies, cold desserts including jellies, créme caramels, and frozendesserts; instant foods such as instant soups and instant soy-beansoups; microwavable foods; and the like. Further, the examples alsoinclude health foods and beverages prepared in the forms of powders,granules, tablets, capsules, liquids, pastes, and jellies. Thecomposition of the present invention can be used for animals, includinghumans. The animals, other than humans, are not particularly limited,and the composition can be used for various livestock, poultry, pets,experimental animals, and the like. Specific examples of the animalsinclude pigs, cattle, horses, sheep, goats, chickens, wild ducks,ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats,monkeys, and the like, but the animals are not limited thereto.

Without wishing to be bound by theory, individuals in whom bacteriabelonging to the group Firmicutes (the group to which the Clostridiumclusters IV and XIVa belong) are relatively abundant gain more bodyweight than individuals in whom bacteria belonging to the groupBacteroidetes are relatively abundant is large. The bacterialcomposition is capable of conditioning absorption of nutrients andimproving feed efficiency. From such a viewpoint, the bacterialcomposition can be used for promoting body weight gain, or for a highefficiency animal feed. Diseases and conditions that would benefit frombody weight gain include, e.g., starvation, cancer, AIDS,gastrointestinal disorders (e.g., celiac disease, peptic ulcer,inflammatory bowel disease (Crohns' disease and ulcerative colitis),pancreatitis, gastritis, diarrhea), hyperthyroidism, infection, renaldisease, cardiac disease, pulmonary disease, connective tissue disease,weight loss caused by medications, anorexia, Addison's disease,dementia, depression, hypercalcemia, Parkinson's disease andtuberculosis.

The addition of the bacterial composition to an antibiotic-free animalfeed makes it possible to increase the body weight of an animal thatingests the animal feed to a level equal to or higher than that achievedby animal ingesting antibiotic-containing animal feeds, and also makesit possible to reduce pathogenic bacteria in the stomach to a levelequal to those in animals consuming typical antibiotic-containing animalfeeds. The bacterial composition can be used as a component of an animalfeed that does not need the addition of antibiotics.

In addition, unlike conventional bacteria (Lactobacillus andBifidobacteria) in commercial use, which are not easy to incorporateinto the livestock production, the present bacterial composition inspore form can be pelletized, sprayed, or easily mixed with an animalfeed and can also be added to drinking water.

Animal feed comprising the bacterial composition can be fed to a widevariety of types of animals and animals of a varying ages and can be fedat regular intervals or for a certain period (for example, at birth,during weaning, or when the animal is relocated or shipped).

The bacterial composition can be used to promote weight gain and enhanceenergy absorption in humans and nonhumans (e.g., farm or other foodanimals).

The bacterial active components of the bacterial composition can bemanufactured using fermentation techniques well known in the art. In oneembodiment, the active ingredients are manufactured using anaerobicfermentors, which can support the rapid growth of bacterial speciesbelonging to the Clostridia class. The anaerobic fermentors may be, forexample, stirred tank reactors or disposable wave bioreactors. Culturemedia such as BL media and EG media, or similar versions of these mediadevoid of animal components can be used to support the growth of thebacterial species. The bacterial product can be purified andconcentrated from the fermentation broth by traditional techniques, suchas centrifugation and filtration, and can optionally be dried andlyophilized by techniques well known in the art.

A food or beverage comprising a bacterial composition described hereincan be manufactured by manufacturing techniques well known in thetechnical field. One or more components (for example, a nutrient) whichare effective for the improvement of an immune function by animmunosuppressive effect may be added to the food or beverage. Inaddition, the food or beverage may be combined with another component oranother functional food exhibiting a function other than the function ofthe improvement of an immune function to thereby serve as amulti-functional food or beverage.

Moreover, the bacterial composition can be incorporated into foodsrequiring a processing step which may destroy ordinary probioticstrains. Specifically, most commercially usable probiotic strains cannotbe incorporated into foods that need to be processed, for example, byheat treatment, long term storage, freezing, mechanical stress, orhigh-pressure treatment (for example, extrusion forming or rollforming). On the other hand, because of the advantageous nature offorming spores, the bacterial composition described herein can be easilyincorporated into such processed foods. For example, the bacterialcomposition in the form of spores can survive even in a dried food, andcan remain living even after being ingested. The bacterial compositioncan withstand low-temperature sterilization processes, typicallyprocesses carried out at a temperature from about 70° C. to about 100°C., both inclusive. The bacterial composition can be incorporated intodairy products that require a pasteurization step. Furthermore, thebacterial composition can withstand long-term storage of many years;high-temperature processing such as baking and boiling; low-temperatureprocessing such as freezing and cold storage; and high-pressuretreatments such as extrusion forming and roll forming.

Many types of foods that need to be processed under such harshconditions include foods which need to be processed in a microwave ovento be edible (for example, oatmeal), foods which need to be baked to beedible (for example, a muffin), foods which need to be subjected to asterilization high-temperature treatment for a short period of time tobe edible (for example, milk), and foods which need to be heated to bedrinkable (for example, hot tea).

The amount of the bacterial composition to be administered or ingestedcan be determined empirically, taking into consideration such factors asthe age, body weight, gender, symptoms, health conditions, of anindividual who will receive it, as well as the kind of bacterialcomposition (a pharmaceutical product, a food or beverage) to beadministered or ingested. For example, the amount per administration oringestion is generally 0.01 mg/kg body weight to 100 mg/kg body weight,and, in specific embodiments, 1 mg/kg body weight to 10 mg/kg bodyweight. Also described herein is a method for suppressing the immunity(reducing the immune response) of a subject, the method beingcharacterized in that the bacteria belonging to the Clostridia class orthe physiologically active substance derived from the bacteria isadministered to or ingested by the subject as described above.

The bacterial composition may be administered to an individual once, orit may be administered more than once. If the composition isadministered more than once, it can be administered on a regular basis(for example, once a day, once every two days, once a week, once everytwo weeks, once a month, once every 6 months, or once a year) or on anas needed or irregular basis. The appropriate frequency ofadministration (which may depend on host genetics, age, gender, andhealth or disease status of the subject, among other factors) may bedetermined empirically. For example, a patient can be administered onedose of the composition, and the levels of the bacterial strains of thecomposition in fecal samples obtained from the patient can be measuredat different times (for example after 1 day, after 2 days, after 1 week,after 2 weeks, after 1 month). When the levels of the bacteria fall to,for example, one half of their maximum value, a second dose can beadministered, and so on.

A product comprising the bacterial composition (a pharmaceuticalproduct, a food or beverage, or a reagent) or a manual thereof may beaccompanied by document or statement explaining that the product can beused to suppress the immunity (including a statement that the producthas an immunosuppressive effect and a statement that the product has aneffect of suppressing the proliferation or function of effectorT-cells). Here, the “provision to the product or the manual thereof withthe note” means that the document or statement is provided to a mainbody, a container, a package, or the like of the product, or the note isprovided to a manual, a package insert, a leaflet, or other printedmatters, which disclose information on the product.

<Method for Inducing Proliferation or Accumulation of Regulatory TCells>

As described above, and as shown in Examples, administration of thebacterial composition to an individual makes it possible to induceproliferation or accumulation of regulatory T cells in the individual.This provides a method of inducing proliferation or accumulation ofregulatory T cells in an individual, the method comprising:administering, to the individual, at least one substance selected fromthe group consisting of: (a) Clostridium saccharogumia, Clostridiumramosum JCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662; (b) aculture supernatant of at least one (a, one or more) of the bacteriadescribed/listed herein; (c) a physiologically active substance derivedfrom a (one or more, at least one) bacterium described/listed herein; ora combination of any two or three of (a), (b) and (c). The bacterialcomposition is administered (provided) to the individual in sufficientquantity to produce the desired effect of inducing proliferation,accumulation or both proliferation and accumulation of regulatory Tcells. It may be administered to an individual in need of treatment,reduction in the severity of or prevention of at least one diseaseselected from an autoimmune disease, an inflammatory disease, anallergic disease, and an infectious disease.

Note that, the “individual” or “subject” may be in a healthy state or adiseased state. The method may further comprise the optional step ofadministering at least one (a, one or more) antibiotic preceding, or incombination with, the bacterial composition. The antibiotic administeredcan be, for example, one which facilitates recolonization of the gut byGram-positive bacteria of the Clostridia class, such as an antibioticthat reduces Gram-negative bacteria. Examples of such antibioticsinclude aminoglycoside antibiotics (amikacin, gentamicin, kanamycin,neomycin, netilmicin, tobramycin, and paromomycin), cephalosporinantibiotics (cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime,cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, and cefoxotin), sulfonamides,ampicillin, and streptomycin.

Moreover, a prebiotic composition such as almond skin, inulin,oligofructose, raffinose, lactulose, pectin, hemicellulose (such asxyloglucan and alpha-glucans), amylopectin, and resistant starch whichare not decomposed in the upper gastrointestinal tract and promote thegrowth of intestinal microbes in the intestinal tract, as well as growthfactors such as acetyl-Co A, biotin, beet molasses, and yeast extracts,preferentially contributes to the proliferation of the bacterial speciesin the composition belonging to the Clostridia class. A method ofinducing proliferation and/or accumulation of regulatory T cells in anindividual can comprise administering, to the individual, at least onesubstance selected from the above in combination with the bacterialcomposition. Also contemplated herein is a composition comprising thebacterial composition and a prebiotic composition.

The above-described antibiotic, and the above-described prebioticcomposition or growth factor may be used in combination. Moreover, atherapeutic composition may be administered to an individual togetherwith at least one substance selected from the group consisting of thebacterial composition, an antibiotic, and a prebiotic composition orgrowth factor.

A therapeutic composition can be, for example, one therapeuticcomposition selected from the group consisting of corticosteroids,mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives,immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine,prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine,theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugsfor rhinitis, anti-cholinergic decongestants, mast-cell stabilizers,monoclonal anti-IgE antibodies, vaccines (preferably, vaccines used forvaccination where the amount of an allergen is gradually increased),anti-TNF inhibitors such as infliximab, adalimumab, certolizumab pegol,golimumab, or etanercept, and combinations thereof. These therapeuticcompositions can be administered prior to, in combination with orfollowing administration of the bacterial composition and optionally,also in combination with an antibiotic, a prebiotic composition, agrowth factor or any combination of an antibiotic, a prebioticcomposition and a growth factor.

There is no particular limitation imposed on the combined use of thetherapeutic composition with at least one substance selected from thegroup consisting of the bacterial composition, the “antibiotic”, and the“prebiotic composition or growth factor”. For example, the “onesubstance” and the therapeutic composition are administered orally orparenterally to an individual simultaneously orsequentially/individually at any appropriate time.

Whether administration of the bacterial composition induces theproliferation and/or accumulation of regulatory T cells can bedetermined by using, as an index, increase or reinforcement of at leastone of the following: the number of regulatory T cells, the ratio ofregulatory T cells in the T cell group of the colon, a function ofregulatory T cells, or expression of a marker of regulatory T cells. Aspecific approach is measurement counts or percentage ofFoxp3-expressing Tregs in a patient sample, such as a biopsy or a bloodsample, promotion (enhancement) of IL-10 expression, promotion(enhancement) of CTLA4 expression, promotion (enhancement) of IDOexpression, suppression of IL-4 expression, or colonization of anindividual with the bacterial composition administered as the index ofthe induction of proliferation or accumulation of regulatory T cells.

Methods for detecting such expression include northern blotting, RT-PCR,and dot blotting for detection of gene expression at the transcriptionlevel; ELISA, radioimmunoas says, immunoblotting, immunoprecipitation,and flow cytometry for detection of gene expression at the translationlevel.

Samples that may be used for measuring such an index include tissues andfluids obtained from an individual, such as blood, obtained in a biopsy,and a fecal sample.

<Method for Predicting Response of an Individual to the BacteriaComposition by Monitoring the Individual's Response to Treatment withthe Composition>

Also described is a method in which an amount (e.g. count) or thepercentage of at least one bacterial species selected from the groupconsisting of: Clostridium saccharogumia, Clostridium ramosum JCM1298,Clostridium ramosum, Flavonifractor plautii, Pseudoflavonifractorcapillosus ATCC 29799, Clostridium hathewayi, Clostridiumsaccharolyticum WM1, Bacteroides sp. MANG, Clostridium saccharolyticum,Clostridium scindens, Lachnospiraceae bacterium 5_1_57FAA,Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616, Clostridiumbolteae ATCC BAA-613, cf. Clostridium sp. MLG055, Erysipelotrichaceaebacterium 2_2_44A, Clostridium indolis, Anaerostipes caccae, Clostridiumbolteae, Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662 in a patient's sample (e.g. a colonic biopsy or a fecalsample) is determined. When the percentage or the count of the bacteriaselected from the list above is lower in an individual than a base linevalue obtained by performing a similar determination on a healthyindividual (e.g., an individual who does not have/has not beenidentified as having a disease or condition for which the bacterialcomposition is a potential treatment such as an auto-immune disease, anallergic condition, cancer, organ rejection), it is determined that theindividual is likely to be responsive to the bacterial composition. Thisdetermination can be used, for example, by a clinician to determinewhether an individual or a patient is likely to benefit from treatmentwith the bacterial composition, or to select an individual or a patientfor inclusion in a clinical trial. The clinician can then administer thebacterial composition to the individual or patient based on thedetermination that the individual or patient is likely to benefit fromtreatment. This determination can also be used as a method to monitor anindividual's response to treatment with the bacterial compositionsdescribed, wherein a higher value of the determination after treatmentwith the bacterial composition (compared to a determination beforetreatment) indicates that the individual has responded favorably totreatment (e.g. is a positive indicator of successful colonization andenhanced immunosuppression in the individual). Optionally, the prognosisand monitoring methods described here may further comprise the step ofmeasuring in the individual's samples the percentages or absolute countsof other commensal species belonging to Clostridium Clusters IV and XIVathat are not present in the bacterial composition, wherein lower thanbaseline values before treatment indicate a higher likelihood of apositive response to treatment, and wherein an increased value aftertreatment indicates that the individual has responded favorably totreatment. In the prognosis and monitoring methods described here, avariety of known methods can be used for determining the composition ofthe microbiota. For example, 16S rRNA sequencing can be used

<Vaccine Adjuvant Composition and Method for Treating or PreventingInfectious Disease or Autoimmune Disease by Using the VaccineComposition>

As described above, and as shown in the Examples, the induction of Tregcells in the colon by bacteria belonging to the Clostridia class has animportant role in local and systemic immune responses. The bacterialcomposition can also be used as an adjuvant to improve the efficacy of avaccine formulation. In one embodiment, the bacterial composition can beused as an adjuvant to a vaccine for the prophylaxis or treatment of anautoimmune disease or an allergic disease (for example, as an adjuvantfor a vaccination protocol where the amount of an allergen is graduallyincreased).

Example of autoimmune diseases and allergic diseases include thosedescribed as the “specific examples of target diseases” in <CompositionHaving Effect of Inducing Proliferation or Accumulation of Regulatory Tcells>.

Other Embodiment

The bacterial composition can also be administered to an individual whois also receiving antibiotic treatment. The present inventors havedemonstrated that antibiotics that act against Gram+bacteria, such asvancomycin or metronidazole, can effectively eliminate or greatly reducebacterial species belonging to the Clostridia class from thegastrointestinal tract of mammals and subsequently decrease the levelsof regulatory T cells (Example 5, FIG. 30). Without wishing to be boundby theory, the key role of bacteria belonging to the Clostridia class inpreserving immune tolerance strongly indicates that their absence orreduced levels can play a key role in autoimmune diseases characterizedby failures of immune tolerance. Accordingly, individuals undergoingcourses of antibiotics against Gram+ bacteria (for example, individualsbeing treated for infections with pathogens such as C. difficile andGiardia), who are at a high risk of experiencing a loss of the bacteriabelonging to the Clostridia class and thus experience immune tolerancedeficits, can be preventively “repopulated” through use of the bacterialcomposition. The bacterial composition can be administered before,simultaneously with, or after the antibiotic treatment, but preferablyit is administered simultaneously or after the antibiotic treatment. Thebacterial composition is preferably administered in spore form, toimprove its resistance to residual antibiotics. Antibiotics againstGram-positive bacteria include, but are not limited to, vancomycin,metronidazole, linezolid, ramoplanin, fidaxomicin, cephalosporinantibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin,cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole);fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox,and norflox); tetracycline antibiotics (tetracycline, minocycline,oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin,ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); and carbapenem antibiotics (ertapenem, doripenem,imipenem/cilastatin, and meropenem).

<Methods to Select Treg-Inducing Organisms>

Also described is a method of obtaining bacteria capable of inducingTregs, comprising (1) isolating the bacterial spore-forming fractionfrom a fecal or biopsy sample obtained from a mammal, preferably a human(e.g. by chloroform treatment or by heat treatment), (2) optionally,orally administering the spore-forming fraction to a non-human mammal,preferably a germ-free non-human mammal; (3) optionally, obtaining afecal sample from the non-human mammal, diluting the fecal sample (forexample diluting it by volume by a factor of 10, 100, 1,000, or 10,000),thereby producing a diluted fecal sample, and orally administering thediluted sample to a second germ-free non-human mammal, wherein optionalstep (3) can be repeated more than one time, (4) plating serialdilutions, under aerobic condition or strictly anaerobic conditions, ofeither the spore-forming fraction obtained in (1) or a sample ofintestinal contents of the non-human mammal of (3), and (5) picking asingle colony from the culture plate. The colony can be further assessedfor the ability of bacteria to induce proliferation of regulatory Tcells and/or accumulation of regulatory T cells using known methods,such as those described in the examples.

Following are examples, which describe specific aspects. They are notintended to be limiting in any way.

Note that mice used in Examples were prepared or produced as follows. Inthe following description, mice may be referred to as “SPF” or “GF”.These “SPF” and “GF” indicate that the mice were maintained in theabsence of specific pathogenic bacteria (specific pathogen-free, SPF),and that the mice were maintained under Germ-Free (GF) conditions,respectively.

<Mice>

C57BL/6, Balb/c, and IQI mice maintained under SPF or GF conditions werepurchased from Sankyo Labo Service Corporation, Inc. (Japan), JAPAN SLC,INC. (Japan), CLEA Japan, Inc. (Japan), or The Jackson Laboratory (USA).GF mice and gnotobiotic mice were bred and maintained within thegnotobiotic facility of The University of Tokyo, Yakult CentralInstitute for Microbiological Research, or Sankyo Labo ServiceCorporation, Inc. Myd88^(−/−), Rip2^(−/−), and Card9^(−/−) mice wereproduced as described in NPL 1 to 3, and backcrossed for 8 generationsor more, so that a C57BL/6 genetic background was achieved. Foxp3^(eGFP)mice were purchased from the Jackson Laboratory.

<Il10^(venus) Mice>

To form a bicistronic locus encoding both Il10 and Venus under controlof an Il10 promoter, a targeting construct was first created.Specifically, a cassette (IRES-Venus-SV40 polyA signal cassette, referto Non-Patent Document 4) which was made of an internal ribosome entrysite (IRES), a yellow fluorescent protein (Venus), and a SV40 polyAsignal (SV40 polyA) and which was arranged next to a neomycin-resistantgene (neo), was inserted between a stop codon and a polyA signal (Exon5) of a Il10 gene. Next, the obtained targeting construct was used tocause homologous recombination with the Il10 gene region in the genomeof mice. Thus, Il10^(venus) mice having an Il10^(venus) alleles wereproduced (refer to FIG. 1). Note that in FIG. 1 “tk” represents a genecoding thymidine kinase, “neo” represents the neomycin-resistant gene,and “BamH1” represents a cleavage site by the restriction enzyme BamH1.

Genomic DNAs were extracted from the Il10^(venus) mice, treated withBamH1, and Southern blotted by use of a probe shown in FIG. 1. FIG. 2shows the obtained results. Wild-type and Il10^(venus) alleles weredetected as bands having sizes of 19 kb and 5.5 kb, respectively. Hence,as is apparent from the results obtained, the homologous recombinationoccurred in the genome of the Il10^(venus) mice.

Further, CD4⁺ Venus⁻ cells or CD4⁺ Venus⁺ cells in the colonic laminapropria of the Il10^(venus) mice were sorted by use of a FACSAria. Then,real-time RT-PCR was carried out on an ABI 7300 system by a method to bedescribed later, to determine the amount of IL-10 mRNA expressed. It wasfound that, since the development of the IL-10 mRNA was detected only inthe CD4⁺ Venus⁺ cells, the expression of IL-10 mRNA in the Il10^(venus)mice was correctly reflected in the expression of Venus. Note that thegerm-free states of such Il10^(venus) mice were established in CentralInstitute for Experimental Animals (Kawasaki, Japan). The Il10^(venus)mice in the germ-free states were maintained in vinyl isolators inSankyo Labo Service Corporation, Inc. (Tokyo, Japan), and used in thefollowing Examples.

Experiments and analyses in Examples were carried out as follows.

<Method for Colonization of Mice with Murine Bacteria and AnalysisThereof>

According to the description in NPL 5 and 6, mice in which SFB orClostridium were colonized were produced. Cecal contents or feces of theobtained gnotobiotic mice were dissolved in sterile water or ananaerobic dilution solution. The dissolved cecal contents or feces asthey were or after a chloroform treatment were orally administered to GFmice. Three strains of the Lactobacillus and 16 strains of theBacteroides were cultured separately from each other in a BL or EG agarmedium in an anaerobic manner. The cultured bacteria were harvested,suspended in an anaerobic TS broth, and orally administrated forcibly toGF mice. The state of the colonization of the bacteria in the mice wasassessed by microscopic observation conducted on a smear preparation offecal pellets.

<Isolation of Intestinal Lamina Propria Lymphocytes and Flow Cytometry>

The small intestine and colon were collected and opened longitudinally.The cecum was also isolated and cecal content was directly frozen at−80° C. or suspended in 2 ml PBS, then added 40% glycerol (finalconcentration 20%), snap-frozen in liquid nitrogen and stored at −80° C.until use. The colon and small intestine were washed in PBS to removeall luminal contents and shaken in Hanks' balanced salt solution (HBSS)containing 5 mM EDTA for 20 min at 37° C. After removing epithelialcells, muscle layers and fat tissue using tweezers, the lamina proprialayers were cut into small pieces and incubated with RPMI1640 containing4% fetal bovine serum, 1 mg/ml collagenase D, 0.5 mg/ml dispase and 40μg/ml DNase I (all Roche Diagnostics) for 1 h at 37° C. in a shakingwater bath. The digested tissues were washed with HBSS containing 5 mMEDTA, resuspended in 5 ml of 40% Percoll (GE Healthcare) and overlaid on2.5 ml of 80% Percoll in a 15-ml Falcon tube. Percoll gradientseparation was performed by centrifugation at 800 g for 20 min at 25° C.The lamina propria lymphocytes were collected from the interface ofPercoll gradient and suspended in ice-cold PBS. For analysis ofregulatory T cells, isolated lymphocytes were labeled with the LIVE/DEADfixable violet dead cell stain kit (Invitrogen) to exclude dead cells inthe analysis. The cells were washed with staining buffer containing PBS,2% FBS, 2 mM EDTA and 0.09% NaN3 and stained surface CD4 withPECy7-labeled anti-CD4 Ab (RM4-5, BD Biosciences). Intracellularstaining of Foxp3 and Helios was performed using the Alexa700-labeledanti-Foxp3 Ab (FJK-16s, eBioscience), Alexa647-labeled anti-Helios(22F6, eBioscience) and Foxp3 Staining Buffer Set (eBioscience). Foranalysis of Th1 and Th17 cells, isolated lymphocytes were stimulated for4 hours with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1μg/ml ionomycin (Sigma) in the presence of GolgiStop (BD Biosciences).After incubation for 4 hours, cells were washed in PBS, labeled with theLIVE/DEAD fixable violet dead cell stain kit and stained surface CD4with PECy7-labeled anti-CD4 Ab. Cells were washed, fixed inCytofix/Cytoperm, permeabilized with Perm/Wash buffer (BD Biosciences),and stained with the APC-labeled anti-IL-17 Ab (eBio17B7, eBioscience)and FITC-labeled anti-IFN-γ Ab (XMG1.2, BD Biosciences). The Ab stainedcells were analyzed with a LSR Fortessa (BD Biosciences), and data wereanalyzed using Flow Jo software (Treestar).

<Real-Time RT-PCR>

From an RNA prepared by using RNeasy Mini Kit (Qiagen), a cDNA wassynthesized by use of a MMV reverse transcriptase (Promega KK). The cDNAobtained was analyzed by real-time RT-PCR using Power SYBR Green PCRMaster Mix (Applied Biosystems) and ABI 7300 real time PCR system(Applied Biosystems), or real-time RT-PCR using SYBR Premix Ex Taq(TAKARA) and Light Cycler 480. For each sample, a value obtained wasnormalized for the amount of GAPDH. A primer set was designed by usingPrimer Express Version 3.0 (Applied Biosystems), and those exhibiting a90% or higher sequence identity at an initial evaluation were selected.The primer set used was as follows:

Foxp3 (SEQ ID NO: 1) 5′-GGCAATAGTTCCTTCCCAGAGTT-3′ (SEQ ID NO: 2)5′-GGGTCGCATATTGTGGTACTTG-3′ CTLA4 (SEQ ID NO: 3)5′-CCTTTTGTAGCCCTGCTCACTCT-3′ (SEQ ID NO: 4) 5′-GGGTCACCTGTATGGCTTCAG-3′GITR (SEQ ID NO: 5) 5′-TCAGTGCAAGATCTGCAAGCA-3′ (SEQ ID NO: 6)5′-ACACCGGAAGCCAAACACA-3′ IL-10 (SEQ ID NO: 7)5′-GATTTTAATAAGCTCCAAGACCAAGGT-3′ (SEQ ID NO: 8)5′-CTTCTATGCAGTTGATGAAGATGTCAA-3′ GAPDH (SEQ ID NO: 9)5′-CCTCGTCCCGTAGACAAAATG-3′ (SEQ ID NO: 10) 5′-TCTCCACTTTGCCACTGCAA-3′Mmp2 (SEQ ID NO: 11) 5′-GGACATTGTCTTTGATGGCA-3′ (SEQ ID NO: 12)5′-CTTGTCACGTGGTGTCACTG-3′ Mmp9 (SEQ ID NO: 13)5′-TCTCTGGACGTCAAATGTGG-3′ (SEQ ID NO: 14) 5′-GCTGAACAGCAGAGCCTTC-3′Mmp13 (SEQ ID NO: 15) 5′-AGGTCTGGATCACTCCAAGG-3′ (SEQ ID NO: 16)5′-TCGCCTGGACCATAAAGAA-3′ Ido1 (SEQ ID NO: 17)5′-AGAGGATGCGTGACTTTGTG-3′ (SEQ ID NO: 18) 5′-ATACAGCAGACCTTCTGGCA-3′.

<Preparation and Culturing of Large Intestinal Epithelial Cells (IECs)>

First, the colon was collected, cut open longitudinally, and rinsed withPBS. Subsequently, the colon was treated with 1 mM dithiothreitol (DTT)at 37° C. for 30 minutes on a shaker, and then vortexed for one minuteto disrupt the epithelial integrity. The released intestinal epithelialcells (IECs) were collected, and suspended in 5 ml of 20% percoll. Thesuspension was overlayered on 2.5 ml of 80% percoll in a 15-ml Falcontube. Then, the tube was centrifuged at 25° C. and 780 g for 20 minutesto conduct cell separation by percoll density gradient centrifugation.Cells at the interface were collected, and used as colonic IECs (purity:90% or higher, viability: 95%). The IECs obtained collected weresuspended in RPMI containing 10% FBS, and 1×10⁵ cells of the IECs werecultured in a 24-well plate for 24 hours. Thereafter, the culturesupernatant was collected, and measured for active TGF-β1 level by ELISA(Promega).

Meanwhile, for culturing T cells in vitro, 1.5×10⁵ MACS-purified splenicCD4⁺ T cells were cultured in each well of a round-bottomed 96-wellplate, together with a 50% conditioned medium in which IECs isolatedfrom GF mice or Clostridium-colonized mice were cultured, and with 25ng/ml of hIL-2 (Peprotech), in the presence or absence of 25 μg/ml of ananti-TGF-β antibody (R&D). Note that 10 μg/ml of an anti-CD3 antibodyand an anti-CD28 antibody (BD Bioscience) were bound to theround-bottomed plate. After a 5-day culture, the CD4⁺ T cells werecollected, and subjected to a real-time PCR.

<Colitis Experimental Model>

A fecal suspension from Clostridium-colonized mice was orallyadministered to C57BL/6 mice (2-week old), which were grown in aconventional environment for six weeks.

For preparing a DSS-induced colitis model, 2% (wt/vol) DSS (reagentgrade, DSS salt, molecular weight=36 to 50 kD, manufactured by MPBiomedicals), together with drinking water, was given to the mice forsix days.

Meanwhile, for preparing an oxazolone-induced colitis model, the micewere presensitized by transdermally applying, onto the mice, 150 μl of a3% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one,Sigma-Aldrich)/100% ethanol solution. Five days after that, 150 μl of a1% oxazolone/50% ethanol solution was intrarectally administered againto the presensitized mice under a light anesthesia. Note that theintrarectal administration was conducted by using a 3.5F catheter.

Each mouse was analyzed daily for body weight, occult blood, bleedingvisible with the naked eyes (gross blood), and the hardness of stool.Moreover, the body weight loss percentage, intestinal bleeding (nobleeding, occult blood (hemoccult+), or bleeding visible with the nakedeyes), and the hardness of stool (normal stool, loose stool, ordiarrhea) were evaluated numerically, and the disease activity index(DAI) was calculated in accordance with the description in “S. Wirtz, C.Neufert, B. Weigmann, M. F. Neurath, Nat Protoc 2, 541 (2007).”

<OVA Specific IgE Reaction>

BALB/c SPF mice were inoculated with a fecal suspension fromClostridium-colonized mice (2-week old), and grown in a conventionalenvironment. Then, 1 μg of OVA (grade V, Sigma) and 2 mg of alum (ThermoScientific), 0.2 ml in total, were intraperitoneally injected to themice (at their ages of 4 weeks and 6 weeks). Sera were collected everyweek from the mice at the root of their tail, and OVA-specific IgE wasmeasured by ELISA (Chondrex). Then, at their ages of 8 weeks, spleniccells were collected, inoculated in a 96-well plate at 1×10⁶ cells perwell, and stimulated with OVA (100 μg/ml) for three days. Thereafter,the culture supernatant was collected, and measured for IL-4 and IL-10levels by ELISA (R&D).

<Statistical Analysis>

The difference between control and experimental groups was evaluated bythe Student's t-test.

<Chloroform Treatment and Oral Inoculation with Fecal Samples Into GFMice>

Human stool (2 g) from a healthy volunteer (Japanese, male, 29 y old)was suspended with 20 ml phosphate-buffered saline (PBS) and passedthrough a 70 μm cell strainer to eliminate clumps and debris. Then fecalsuspension was mixed with or without chloroform (final concentration3%), and incubated in a shaking water bath for 60 min. The fecalsuspensions without chloroform treatment were orally inoculated intogerm-free (GF) mice (250 μl/mouse). After evaporation of chloroform bybubbling with N2 gas for 30 min, the aliquots containingchloroform-resistant (spore-forming) fraction of human intestinalbacteria were inoculated into IQI GF mice. Each group of ex-GF mice wasseparately kept in a vinyl isolator for 3 or 4 weeks.

<Co-Housing Experiment>

To evaluate whether Treg-inducing human bacteria can be transmittedhorizontally, IQI GF mice were co-housed for 4 weeks with ex-GF micecolonized with chloroform-treated human feces (Example 21 mice) in avinyl isolator (6 mice, designated as mouse #D1 to #D6

<Inoculation with Diluted Cecal Contents into GF Mice>

The frozen cecal content from ex-GF mice inoculated withchloroform-treated human feces (#C4) was suspended in 10 times volume(w/v) of PBS, passed through a 70 μm cell strainer and treated 3%chloroform. Then the suspension was diluted 2000 (for 4 mice, designatedas mouse #E1 to #E4) or 20000 (for 8 mice, designated as mouse #F1 to#F8)-fold with PBS and orally inoculated into GF IQI mice (2.5×10⁵ or2.5×10⁴ cells/250 μl/mouse). After 4 weeks, lymphocytes were collectedfrom colon and small intestine and analyzed for Foxp3+ Treg cellproportion and their Helios expression. Cecal contents were frozen andstored at −80° C. until use.

<Re-Colonization Experiments>

The frozen cecal content from ex-GF mice inoculated with 20000-folddilution (#F3, 7 and 8) was suspended in 10 times volume (w/v) of PBS,passed through a 70 μm cell strainer and treated 3% chloroform. Thesuspensions were orally inoculated into GF IQI mice (5, 4 or 4 mice;designated as mouse #G1 to #G5, #H1 to #H4 or #I1 to #I4, respectively).After 4 weeks, colon and small intestine were collected and analyzed forFoxp3+ Treg cell proportion and their Helios expression. Cecal contentswere suspended in 20% glycerol solution, snap-frozen in liquid nitrogenand stored at −80° C.

<Cultured Bacteria-Colonization Experiments>

The glycerol stock of cecal content from #G2 mouse was diluted with PBSand seeded onto BL agar plate. After 48 hours, all bacterial colonieswere collected by scraping the plates with a plate scraper andinoculated into GF IQI mice (4 mice, designated as mouse #K1 to #K 4).Six bacterial strains were isolated from the freeze stock of cecalcontent from #F8 mouse using BL agar plate. These isolated strains wereinoculated into GF IQI mice (4 mice, designated as mouse #J1 to #J4).(Details of the culture method are described below.)

<16S rRNA Gene Quantitative PCR Analysis

Using a QIAamp DNA Stool mini kit (QIAGEN), bacterial genomic DNA wasisolated from the human stool from a healthy volunteer as describedabove (human stool), cecal contents from GF mice gavaged withchloroform-treated human stool (cecal content of B-4 mouse) or fecesfrom SPF ICR mouse (feces of SPF mouse). The isolated DNA was used astemplate for quantitative PCR. The amplification program consisted ofone cycle at 95° C. for 1 min, followed by 50 cycles at 95° C. for 10 sand 60° C. for 30 s. Quantitative PCR analysis was carried out using aLightCycler 480 (Roche). Relative quantity was calculated by the ΔCtmethod and normalized to the amount of total bacteria. The followingprimer sets were used: total bacteria, 5′-GGTGAATACGTTCCCGG-3′(SEQ IDNO.: 45) and 5′-TACGGCTACCTTGTTACGACTT-3′(SEQ ID NO.: 46); Clostridiumcluster XIVa (Clostridium coccoides subgroup),5′-AAATGACGGTACCTGACTAA-3′ (SEQ ID NO.: 47) and5′-CTTTGAGTTTCATTCTTGCGAA-3′(SEQ ID NO.: 48); Clostridium cluster IV(Clostridium leptum) 5′-CCTTCCGTGCCGSAGTTA-3′(SEQ ID NO.: 49) and5′-GAATTA AACCACATACTCCACTGCTT-3′(SEQ ID NO.: 50); Bacteroides,5′-GAGAGGAAGGTCCCCCAC-3′(SEQ ID NO.: 51) and5′-CGCTACTTGGCTGGTTCAG-3′(SEQ ID NO.: 52); Bifidobacterium,5′-CGGGTGAGTAATGCGTGACC-3′ (SEQ ID NO.: 53) and5′-TGATAGGACGCGACCCCA-3′(SEQ ID NO.: 54). Note that mice gavaged withchloroform-treated human stool exhibited high levels of spore-formingbacteria, such as Clostridium clusters XIVa and IV, and a severedecrease of non-spore-forming bacteria, such as Bacteroides andBifidobacterium, compared with the human stool before chloroformtreatment.

<Isolation of DNA from Cecal Contents for 16S rRNA Gene MetasequenceAnalysis>

The cecal contents of A1-1, A2-4, B-4, E-3, E-7, E-8, F-2, G-3, H-3, 1-3and J-3 were collected by centrifugation at 5000×g for 10 min at 4° C.,suspended in 10 ml of Tris-EDTA containing 10 mM Tris-HCl and 1 mM EDTA(pH 8), and then used for DNA isolation. Lysozyme (SIGMA, 15 mg/ml) wasadded to the cell suspension. After incubation at 37° C. for 1 h withgentle mixing, a purified achromopeptidase (Wako) was added (final 2000unit/ml) and incubated at 37° C. for 30 min. Then, sodium dodecylsulfate (final 1%) was added to the cell suspension and mixed well.Subsequently, proteinase K (Merck) was added (final 1 mg/ml) to thesuspension and the mixture was incubated at 55° C. for 1 h.High-molecular-weight DNA was isolated and purified by phenol/chloroformextraction, ethanol, and finally polyethyleneglycol precipitation.

<16S rRNA Gene Metasequence>

An aliquot of the DNA was used for PCR amplification and sequencing ofbacterial 16S rRNA genes. ˜330 bp amplicons, spanning variable region1-2 (V1-2) of the gene were generated by using (i) modified primer 8F(5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG+B arcode+agrgtttgatymtggctcag-3′ (SEQID NO.: 55)) which consists of 454 adaptor sequence (underlined), asample specific, error correcting barcode (10 bases, bold) and theuniversal bacterial primer 8F and (ii) modified primer 338R(5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG+tgctgcctcccgtaggagt-3′(SEQ ID NO.:56)) which contains 454 adaptor sequence (underlined) and the bacterialprimer 338R. Polymerase chain reactions were performed for each fecalDNA sample: each 50-μL reaction contained 40 ng of DNA, 5 μl of 10×ExTaq buffer (TAKARA), 5 μl of 2.5 mM dNTP mixture, 0.2 μl Ex Taq and 0.2μM of each primer. PCR conditions consisted of an initial denaturationstep performed at 96° C. for 2 min, followed by 20 cycles ofdenaturation (96° C., 30 s), annealing (55° C., 45 s) and amplification(72° C., 1 min) and final amplification step performed at 72° C. for 10min. Amplicons generated from each sample were subsequently purifiedusing AMPur XP (Beckman Coulter). The amount of DNA was quantified usingQuant-iT Picogreen dsDNA Assay Kit (Invitrogen) and TBS-380miniFluorometer (Turner Biosystems). The amplified DNA were used as templatefor 454 GS Junior (Roche) pyrosequencing. The sequences were performedusing GS Junior Titanium emPCR Kit-Lib-L, GS Junior Titanium SequencingKit and GS Junior Titanium PicoTiterPlate Kit (all Roche) according tothe manufacturer's manuals (GS Junior Titanium Series, emPCRAmplification Method Manual—Lib-L and Sequencing Method Manual).Resulting sequences (3400 reads were produced for each sample) wereclassified into OTU on the basis of sequence similarity (>97% identity).Representative sequences from each OTU were compared with sequences innucleic acid databases (Ribosomal Database Project) using BLAST todetermine the closest relatives. Then, OTUs were classified into specieson the basis of the closest relatives. All data of close relatives andthe number of reads are shown in Table. 1.

<Isolation of Bacterial Strains>

Bacterial strains were isolated from the cecal contents of #F8, #G2, #I1and #K3 by plating serial dilutions of the cecal samples under aerobiccondition or strictly anaerobic conditions (80% N2 10% H2 10% CO2) ontoBL agar (Eiken Chemical) or EG agar plates containing medium with thefollowing components (quantities expressed per liter): Meat extract 500ml; Proteose peptone No. 3 (10.0 g, Difco); Yeast Extract (5.0 g,Difco); Na2HPO4 (4.0 g); D(+)-Glucose (1.5 g); Soluble Starch (0.5 g);L-cystine (0.2 g), L-cysteine-HCl—H2O (0.5 g); Tween80 (0.5 g); BactoAgar (16.0 g, Difco); defibrinated horse blood (50 ml). After culture at37° C. for 2 or 4 days, each single colony was picked up and culturedfor additional 2 or 4 days at 37° C. by ABCM broth or EG agar plate. Theisolated strains were collected into EG stock medium (10% DMSO) andstored at −80° C. For suspension of isolated strains to re-inoculatemice, TS medium (27.5 g of trypticase soy broth w/o dextrose, 0.84 g ofNa2CO3, 0.5 g of L-cysteine-HCl—H2O, 1000 ml of distilled water, pHadjusted to 7.2+/−0.2 with NaOH, then autoclaved for 15 minutes at 115degrees Celsius). To identify the isolated strains, 16SrRNA coding genesequences were performed. The 16S rRNA genes were amplified bycolony-PCR using KOD FX (TOYOBO), 16S rRNA gene-specific primer pairs:8F (5′-AGAGTTTGATCMTGGCTCAG-3′(SEQ ID NO.: 57)) and 519R(5′-ATTACCGCGGCKGCTG-3′(SEQ ID NO.: 58)) for C. indolis, C. bolteae,Bacteroides sp. MANG, L. bacterium DJF_VP30, A. colihominis,Ruminococcus sp. ID8, C. lavalense, C. symbiosum and E. contortum or1513R (5′-ACGGCTACCTTGTTACGACTT-3′(SEQ ID NO.: 59)) for C.saccharogumia, C. ramosum, F. plautii, C. hathewayi, C. scindens,Clostridium sp. 2335, Clostridium sp. 14616 and cf Clostridium sp.MLG055 and GeneAmp PCR System 9700 (Applied Biosystems). Theamplification program consisted of one cycle at 98° C. for 2 min,followed by 40 cycles at 98° C. for 10 s, 57° C. for 30 s and 68° C. for40 s. Each amplified DNA was purified from the reaction mixture usingIllustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).Sequence analysis was performed using BigDye Terminator V3.1 CycleSequencing Kit (Applied Biosystems) and Applied Biosystems 3730xl DNAanalyzer (Applied Biosystems). The resulting sequences were comparedwith sequences in nucleic acid databases using BLAST to determine theclosest relatives. The closest relatives and % identity of all isolatedstrains, information for genus-species of the closest relatives,Clostridium cluster, ID of mouse from which was derived, maximumsimilarity and culture medium of isolated strains were summarized inTable 2.

Example 1: First, it was investigated whether or not accumulation ofregulatory T cells (Treg cells) in the colonic lamina propria wasdependent on commensal bacteria. Specifically, lymphocytes were isolatedfrom peripheral lymph nodes (pLN) of Balb/c mice bred in the absence ofspecific pathogenic bacteria (SPF) or from lamina propria of the colonor the small intestine (SI) of the mice. The CD4 and Foxp3 were stainedby antibodies. Then, the ratio of Foxp3⁺ cells in CD4⁺ lymphocytes wasanalyzed by flow cytometry. The results showed that Foxp3⁺ Treg cellswere present at a high frequency in the lamina propria of thegastrointestinal tracts, especially in the colonic lamina propria, ofthe mice kept under the environment free from specific pathogenicmicroorganisms (SPF). In addition, it was also found that the number ofthe Foxp3⁺ Treg cells in the colonic lamina propria gradually increasedup to three months after their birth, whereas the number of the Foxp3⁺Treg cells in the peripheral lymph nodes was basically constant from thetime of two weeks after their birth.

Example 2: Next, it was investigated whether or not the temporalaccumulation of the Treg cells in the colon as found in Example 1 had arelationship with the colonization of intestinal commensal microbiota.Specifically, the expression of CD4 and the expression of Foxp3 inlymphocytes isolated from the small intestine, the colon, and theperipheral lymph nodes of mice bred under a germ-free (GF) or SPFenvironment (8 weeks old: Balb/c mice, IQI mice, and C57BL/6 mice) wereanalyzed. Similar results were obtained in three or more independentexperiments.

In addition, lamina propria lymphocytes were collected from SPF mice andGF mice (Balb/c mice or C57BL/6 mice). CD4 and Foxp3 were stained withantibodies. Then, the lamina propria lymphocytes were analyzed by FACS.

Further, lymphocytes were isolated from the lamina propria of the colon,the lamina propria of the small intestine (SI), Peyer's patches (PPs),and mesenteric lymph nodes (MLNs) of mice (SPF C57BL/6 mice) to whichantibiotics were orally administered with water for eight weeks. CD4 andFoxp3 were stained with antibodies. Then, the lymphocytes were analyzedby FACS. Similar results (the ratio of the Foxp3⁺ cells in the CD4⁺cells of an individual mouse) were obtained in two or more independentexperiments. Note that the following antibiotics were used incombination in accordance with the description in the followingdocument:

ampicillin (A; 500 mg/L, Sigma)

vancomycin (V; 500 mg/L, NACALAI TESQUE, INC.)

metronidazole (M; 1 g/L, NACALAI TESQUE, INC.)

neomycin (N; 1 g/L, NACALAI TESQUE, INC.)

-   Rakoff-Nahoum, J. Paglino, F. Eslami-Varzaneh, S. Edberg, R.    Medzhitov, Cell 118, 229 (Jul. 23, 2004)-   Fagarasan et al., Science 298, 1424 (Nov. 15, 2002)

As is apparent from the results the frequencies and the absolute numbersof Foxp3⁺ CD4⁺ cells in the small intestine and the peripheral lymphnodes of the GF mice were equal to or greater than those of the SPFmice. In addition, the numbers of the Treg cells in the small intestinallamina propria, Peyer's patches, and mesenteric lymph nodes of the SPFmice to which the antibiotics were orally administered for eight weekswere equal to or greater than those of the SPF mice that had notreceived antibiotics. Meanwhile, the number of the Foxp3⁺ CD4⁺ cells inthe colonic lamina propria of the GF mice was decreased significantly incomparison with that of the SPF mice. This decrease was commonlyobserved among mice of different genetic backgrounds (Balb/c, IQI, andC57BL/6), as well as among mice bred in different animal facilities. Inaddition, it was also shown that the number of Treg cells in the coloniclamina propria of the SPF C57BL/6 mice to which the antibiotics wereadministered was decreased significantly.

Example 3: Next, it was directly checked whether or not the decrease inthe number of the Treg cells in the colonic lamina propria of the GFmice shown in Example 2 was attributed to the absence of microbiota.Specifically, a fecal suspension of B6 SPF mice purchased from TheJackson Laboratory was orally administered to GF-IQI mice(conventionalization). Three weeks after the administration, lymphocyteswere isolated from the colonic lamina propria, and the expression ofFoxp3 in CD4⁺ lymphocytes was analyzed. The results showed that thenumber of Treg cells in the small intestinal lamina propria did notchange. However, the number of the Treg cells in the colonic laminapropria increased significantly. Hence, it was shown that host-microbialinteraction played an important role in the accumulation of Foxp3⁺ Tregcells in the colonic lamina propria, while the accumulation of the Tregcells in the small intestinal lamina propria had a different mechanism.

Example 4: Next, the relationship between the gut-associated lymphoidtissues of mice and the number of Foxp3⁺ cells in the colonic laminapropria of the mice was investigated in accordance with the methoddescribed in M. N. Kweon et al., J Immunol 174, 4365 (Apr. 1, 2005).Specifically, 100 μg of an extracellular domain recombinant protein (afusion protein (LTβR-Ig) between a lymphotoxin β receptor (LTβR) and aFc region of human IgG1, refer to Honda et al., J Exp Med 193, 621 (Mar.5, 2001)) was injected intraperitoneally into pregnant C57BL/6 mice 14days after conception. The LTβR-Ig was again injected intraperitoneallyinto fetuses obtained from such mice, so that mice from which isolatedlymphoid follicles (ILFs), Peyer's patches (PPs), and colonic-patches(CPs) were completely removed were produced. Then, the ratios of Foxp3⁺cells in CD4⁺ cells in the colonic lamina propria of the mice treatedwith the LTβR-Ig, and mice treated with rat IgG (control) were analyzedby FACS. The results show that the ratio of the Foxp3⁺ cells in thecolonic lamina propria of the mice deficient in isolated lymphoidfollicles, Peyer's patches, and the colonic-patches (the mice treatedwith the LTβR-Ig) rather increased. Accordingly, it was suggested thatthe decrease in the number of the Treg cells in the colonic laminapropria of the GF mice and the mice treated with the antibiotics wascaused because the transmission of specific signals which promotes theaccumulation of Treg cells in the colonic lamina propria and which iscaused by the intestinal microbes did not occur, rather than simplybecause of a secondary effect of disorganized gut-associated lymphoidtissues.

Example 5: To investigate whether or not a specific intestinal florainduced the accumulation of colonic Treg cells, vancomycin as anantibiotic against Gram-positive bacteria or polymyxin B as anantibiotic against Gram-negative bacteria was administered to SPF mice(from 4 weeks of age) for four weeks, and analyzed for the ratio ofFoxp3⁺ cells in the CD4⁺ cell group ([%] Foxp3⁺ in CD4).

The results show that the number of Treg cells in the colon of the miceto which vancomycin was administered was markedly decreased incomparison with that of the control. In contrast, no influence wasobserved on the number of Treg cells of the mice to which polymyxin Bwas administered. Those facts suggested that Gram-positive commensalbacteria played a major role in accumulation of Treg cells.

Example 6: A recent report has suggested that spore-forming bacteriaplay an important role in intestinal T cells response (see V.Gaboriau-Routhiau et al., Immunity 31, 677 (Oct. 16, 2009)). In thisrespect, fecal microorganisms (spore-forming fraction) resistant to 3%chloroform were orally administered to GF mice, which were then analyzedfor the ratio of Foxp3⁺ cells in the CD4⁺ cell group ([%] Foxp3⁺ inCD4).

Three weeks after the administration of the chloroform-treated feces,the number of Treg cells in the administered mice was markedly increasedto the same level as those of the SPF mice and the GF mice to which theuntreated feces was forcibly administered.

Accordingly, considering the results shown in Example 5 in combination,it was revealed that the specific components of the indigenousmicrobiota were highly likely to belong to the Gram-positive group, andthat the spore-forming fraction played an important role in theinduction of Treg cells.

Example 7: Next, the species of the intestinal microbiota which inducedthe accumulation of Treg cells in the colon as suggested in Examples 4to 6 were identified. Specifically, segmented filamentous bacteria(SFB), 16 strains of the Bacteroides spp. (Bactero. (6 strains of B.vulgatus, 7 of the B. acidifaciens group 1, and 3 of the B. acidifaciensgroup 2)), 3 strains of the Lactobacillus (Lacto. (L. acidophilus, L.fermentum, and L. murinum)), and 46 strains of Clostridium spp. (Clost.,refer to “Itoh, K., and Mitsuoka, T. Characterization of clostridiaisolated from faeces of limited flora mice and their effect on caecalsize when associated with germ-free mice. Lab. Animals 19: 111-118(1985))”), or microbiota collected from mice (SPF) bred under aconventional environment was orally administered to GF-Balb/c mice orGF-IQI mice. The mice were maintained in vinyl isolators for threeweeks. Then, CD4 cells were isolated from the colon and the smallintestine of these mice. The numbers of Treg cells in the colon and thesmall intestine were analyzed by flow cytometry.

The bacteria belonging to the genus Clostridium are classified bysequencing of 16S rRNA gene, as follows. Specifically, the 16S rRNAgenes of the bacteria were amplified by PCR using 16S rRNA gene-specificprimer pairs: 5′-AGAGTTTGATCMTGGCTCAG-3′ (SEQ ID NO: 60) and5′-ATTACCGCGGCKGCTG-3′ (SEQ ID NO: 61) (see T. Aebischer et al.,Vaccination prevents Helicobacter pylori-induced alterations of thegastric flora in mice. FEMS Immunol. Med. Microbiol. 46, 221-229(2006)). The 1.5-kb PCR product was then introduced into pCR-BluntVector. The inserts were sequenced and aligned using the ClustalWsoftware program. The resulting sequences of 16S rRNA genes derived fromstrain 1-41 of 46 strains of Clostridium spp. were shown in SEQ ID NO:21-61. A phylogenetic tree was constructed by the neighbor-joiningmethod with the resulting sequences of the 41 strains of Clostridium andthose of known bacteria obtained from Genbank database using Megasoftware.

The results showed no effect on the number of the Treg cells in thecolon was observed in the GF mice in which the segmented filamentousbacteria (SFB) were colonized. Moreover, mice in which the cocktail ofthree strains of Lactobacillus was colonized gave similar results. Onthe other hand, it was shown that the accumulation of Foxp3⁺ cells inthe colonic lamina propria was strongly induced in the mice in which 46strains of Clostridium spp. were colonized. Importantly, suchaccumulation was promoted irrespective of the genetic backgrounds of themice, and led to the increase in number similar to that in the SPF micealthough intestinal microbiota of only a single genus were colonized. Itwas also shown that the colonization of the Clostridium did not changethe number of Treg cells in the small intestinal lamina propria. Notethat, when the 16 strains of Bactericides spp. were colonized, thenumber of Treg cells in the colon was increased significantly. However,the extent of the increase varied depending on the genetic background ofthe mice in which the bacteria were colonized.

Example 8: Next, CD4 expression, Foxp3 expression, and Helios expressionin LP lymphocytes of the thymuses and the colons of SPF mice, GF mice,Lactobacillus-colonized mice, and Clostridium-colonized mice wereanalyzed by flow cytometry.

The results show that most Foxp3⁺ cells found in the SPF mice or theClostridium-colonized mice did not express Helios. Note that Helios is atranscription factor known to be expressed in thymic-derived naturalTreg cells (see A. M. Thornton et al., J Immunol 184, 3433 (Apr. 1,2010)). Accordingly, it was suggested that most of the Treg cells in theSPF mice and the Clostridium-colonized mice were Treg cells induced inperipheral portions (so-called iTreg cells).

Example 9: Next, it was investigated whether or not the colonization ofthe Clostridium or the like had an influence on other T cells.Specifically, SFB, 16 strains of Bacteroides spp. (Bactero.), 46 strainsof Clostridium spp. (Clost.), or microbiota collected from mice bredunder a conventional environment (SPF) was colonized in GF IQI mice.Three weeks later, lymphocytes in the colonic lamina propria wereisolated from these mice, and stimulated with PMA (50 ng/ml) andionomycin (1 μg/ml) for four hours in the presence of Golgistop (BDBioscience). After the stimulation was given, intracellular cytokineswere stained by using an anti-IL-17 PE antibody (TC11-18H10) and ananti-IFN-g FITC antibody (BD Bioscience) in accordance with the manualof a cytofix/cytoperm kit (BD Bioscience). Then, the ratio of IFN-γ⁺cells or IL-17⁺ cells in CD4⁺ leucocytes was analyzed by flow cytometry.The results show that the colonization of the Clostridium did not haveany influence on Th1 cells (CD4⁺ IFN-γ⁺ cells) in the colon, and causedonly a slight increase of Th17 cells (CD4⁺ IL-17⁺ cells). Accordingly,it was suggested that the genus Clostridium was a genus of bacteriawhich specifically induced Treg cells.

Example 10: It has been reported that 46 strains of Clostridium spp.exert an influence on the accumulation of CD8⁺ intestinal tractintraepithelial lymphocytes (IELs) in the colon. Accordingly, it isconceivable that Clostridium regulates the immune system in variousaspects, and that Clostridium exhibits a marked ability to induce andmaintain Treg cells especially in the colon, as described above. Inaddition, a kind of cytokines, transforming growth factor-β (TGF-β), isknown to play an important role in regulation of Treg cell generation.

In this respect, it was examined whether or not the colonization ofClostridium provided a colonic environment rich in TGF-β. Specifically,first, the whole colons of GF mice, Clostridium-colonized mice, andLactobacillus-colonized mice were cultured for 24 hours, and the culturesupernatants thereof were measured for the concentration of active TGF-β(TGF-β1) by ELISA (the number of mice analyzed was four per group).

The results show that the amount of TGF-β produced in the colons of theClostridium-colonized mice was significantly greater than that in colonsof the GF mice and the Lactobacillus-colonized mice.

Next, intestinal epithelial cells (IECs) of GF mice andClostridium-colonized mice were cultured for 24 hours, and the culturesupernatants thereof were measured for the concentration of activeTGF-β(TGF-β1) by ELISA (the number of mice analyzed was four per group).

The results show that TGF-β was detected in the culture supernatant ofthe IECs isolated from the Clostridium-colonized mice, whereas no TGF-βwas detected in the culture supernatant of the IECs isolated from the GFmice.

Next, as described above, splenic CD4⁺ T cells were cultured for fivedays together with a 50% conditioned medium in which IECs isolated fromthe GF mice or the Clostridium-colonized mice were cultured, and withthe anti-CD3 antibody, in the presence or absence of an anti-TGF-βantibody. Then, the T cells were collected, and analyzed for expressionof Foxp3 by real-time RT-PCR.

The results show that when the culture supernatant of the IECs derivedfrom the Clostridium-colonized mice was added to the splenic CD4⁺ Tcells, differentiation into Foxp3-expressing cells was accelerated.Meanwhile, differentiation into Treg cells was inhibited by theanti-TGF-β antibody.

The expression of MMP2, MMP9, and MMP13, which are thought to contributeto the activation of latent TGF-β was investigated. The expression ofindoleamine 2,3-dioxygenase (IDO), which is thought to be involved inthe induction of Treg cells, was also investigated. Specifically, 46bacterial strains of the genus Clostridium (Clost.), or three bacterialstrains of the genus Lactobacillus (Lacto.) were orally administered toC57BL/6 germ-free mice. Three weeks after administration, IECs werecollected, and analyzed for relative mRNA expression levels of MMP2,MMP9, MMP13, and IDO genes by real-time RT-PCR (the number of miceanalyzed was three per group).

For the relationship between the activation of latent TGF-β and theabove-describe MMP, see D'Angelo et al., J. Biol. Chem. 276,11347-11353, 2001; Heidinger et al., Biol. Chem. 387, 69-78, 2006; Yu etal., Genes Dev. i4, 163-176, 2000. For the relationship between IDO andthe induction of Treg cells, see G. Matteoli et al., Gut 59, 595 (May,2010).

The results show in agreement with the production of TGF-β describedabove, that transcription products of the genes encoding MMP2, MMP9, andMMP13 were expressed at higher levels in the IECs derived from theClostridium-colonized mice than in those in the GF mice and in theLactobacillus-colonized mice.

Moreover, IDO was expressed only in the Clostridium-colonized mice.

Accordingly, it was revealed that the Clostridium activated the IECs,and led to the production of TGF-β and other Treg cell-inducingmolecules in the colon.

Example 11: Next, it was investigated whether or not the Treg cellaccumulation induced by the colonization of the Clostridium wasdependent on signal transmission by pathogen-associated molecularpattern recognition receptors. Specifically, the numbers of Treg cellsin the colonic lamina propria of each SPF mice of Myd88^(−/−) (deficientin Myd88 (signaling adaptor for Toll-like receptor)), Rip2^(−/−)(deficient in Rip2 (NOD receptor adaptor)), and Card9^(−/−) (deficientin Card9 (essential signal transmission factor for Dectin-1 signaltransmission)) were examined. In addition, Clostridium spp. were causedto be colonized in the Myd88^(−/−) GF mice, and the change in the numberof Treg cells was investigated. The results show that the number of Tregcells of each kind of the SPF mice deficient in the associated factorsof the pathogen-associated molecular pattern recognition receptors didnot change relative to that of wild-type mice of the same litter, whichserved as a control. In addition, it was found that when Clostridiumspp. were colonized in GF mice deficient in Myd88, the accumulation ofTreg cells in the colonic lamina propria was induced. Accordingly, ithas been suggested that the mechanism of inducing the accumulation ofTreg cells in the colonic lamina propria relies not on activation ofrecognition pathway for major pathogen-associated molecular patterns asis caused by most bacteria, but on specific commensal bacterial species.

Example 12: Intestinal tract Foxp3⁺ Treg cells are known to exert someimmunosuppressive functions through IL-10 production (refer to NPL 9).Meanwhile, animals having CD4⁺ Foxp3⁺ cells from which IL-10 isspecifically removed are known to develop inflammatory bowel disease(refer to NPL 18). In this respect, first, the expression of IL-10 inlymphocytes of various tissues was examined. Specifically, lymphocyteswere isolated from various tissues of SPF Il10^(venus) mice, and theexpression of CD4 and the expression of Venus were analyzed by flowcytometry.

Lymphocytes in the colonic lamina propria were isolated fromIl10^(venus) mice, and the expression of T cell receptor β chain (TCRβ)on the surfaces of the cells was detected by FACS.

Lymphocytes in the colonic lamina propria were isolated fromIl10^(venus) mice. The lymphocytes were stimulated with PMA (50 ng/ml)and ionomycin (1 μg/ml) for four hours in the presence of Golgistop (BDBioscience). Then, after the stimulation was given, intracellularcytokines were stained by using an anti-IL-17 PE antibody, an anti-IL-4APC antibody (11B11), and an anti-IFN-g FITC antibody (BD Bioscience) inaccordance with the manual of a cytofix/cytoperm kit (BD Bioscience).

In addition, Foxp3⁺ CD4⁺ cells and Foxp3⁻ CD4⁺ cells were isolated fromthe spleen (Spl) of Foxp3^(eGFP) reporter mice, and Venus⁺ cells wereisolated from the colonic lamina propria and the small intestine (SI)lamina propria of Il10^(venus) mice. The obtained cells were analyzed interms of expression of predetermined genes. The gene expression wasanalyzed by real-time RT-PCR using a Power SYBR Green PCR Master Mix(Applied Biosystems) and an ABI 7300 real time PCR system (AppliedBiosystems). Here, the value for each cell was normalized for the amountof GAPDH.

The results show that almost no Venus⁺ cells (IL-10-producing cells)were detected in the cervical lymph nodes (peripheral lymph nodes),thymus, peripheral blood, lung, and liver of mice kept under the SPFconditions. Meanwhile, in the spleen, Peyer's patches, and mesentericlymph nodes thereof, Venus⁺ cells were slightly detected. On the otherhand, many Venus⁺ cells were found in the lymphocytes in the smallintestine lamina propria and colonic lamina propria. In addition, mostof the Venus⁺ cells in the intestines were positive for CD4, and alsopositive for T cell receptor β chain (TCRβ). It was found that theVenus⁺ CD4⁺ T cells expressed Foxp3 and other Treg cell-associatedfactors such as a cytotoxic T-Lymphocyte antigen (CTLA-4) and aglucocorticoid-induced TNFR-associated protein (GITR), although theVenus⁺ CD4⁺ T cells showed none of the phenotypes of Th2(IL-4-producing) and Th17 (IL-17-producing). It was shown that theexpression level of CTLA-4 in the intestinal Venus⁺ cells was higherthan that in the splenic GFP⁺ Treg cells isolated from the Foxp3^(eGFP)reporter mice.

Example 13: Venus⁺ cells can be classified into at least two subsets,namely, Venus⁺ Foxp3⁺ double positive (DP) Treg cells and Venus⁺ Foxp3⁻Treg cells on the basis of intracellular Foxp3 expression. Cells of thelatter subset correspond to type 1 regulatory T cells (Tr1) (refer toNPL 8 and 9). In this respect, the Venus⁺ cells (IL-10-producing cells)observed in Example 8 were investigated in terms of the expression ofFoxp3. Specifically, the expression of CD4, Foxp3, and Venus in thelamina propria of the colon and the lamina propria of the smallintestine of Il10^(venus) mice kept under GF or SPF conditions wasanalyzed by FACS, and the numbers of Venus⁺ cells in the intestinaltract lamina propria were compared between SPF and GF Il10^(venus) mice.

In addition, the intracellular expression of Venus and Foxp3 in CD4cells in various tissues of SPF Il10^(venus) mice was analyzed by flowcytometry.

In order to investigate whether or not the presence of commensalbacteria had any influence on the expression of IL-10 in regulatorycells in the gastrointestinal tracts, germ-free (GF) Il10^(venus) micewere prepared. Then, predetermined species of bacteria were caused to becolonized in the obtained GF Il10^(venus) mice. Three weeks after thespecies of bacteria were colonized, a CD4⁺ cell group (V⁺F⁻,Venus⁺Foxp3⁻ cells; V⁺F⁺, Venus⁺Foxp3⁺ cells; and V⁻F⁺, Venus⁻Foxp3⁺cells) in which Foxp3 and/or Venus were expressed in the colon and thesmall intestine was analyzed by flow cytometry.

In order to check whether or not the presence of commensal bacteria hadany influence on the expression of IL-10 in regulatory cells in thegastrointestinal tracts, antibiotics were orally given with water tofive or six Il10^(venus) mice per group for 10 weeks. The followingantibiotics were used in combination.

ampicillin (A; 500 mg/L Sigma)

vancomycin (V; 500 mg/L NACALAI TESQUE, INC.)

metronidazole (M; 1 g/L NACALAI TESQUE, INC.)

neomycin (N; 1 g/L NACALAI TESQUE, INC.)

Then, CD4 and Foxp3 of lymphocytes in the lamina propria of the colon,the lamina propria of the small intestine (SI), mesenteric lymph nodes(MLN), and Peyer's patches (PPs) were stained with antibodies, andanalyzed by FACS. The results were obtained from two or more independentexperiments which gave similar results.

The results show that the small intestinal lamina propria was rich inVenus⁺ Foxp3⁻ cells, namely, Tr1-like cells, and that the Venus⁺ Foxp3⁺DP Treg cells were present at a high frequency in the colon of the SPFmice. In contrast, although sufficient numbers of Foxp3⁺ cells wereobserved also in other tissues, the expression of Venus was not observedin almost all of the cells.

In addition, it was shown that all regulatory T cell fractions of Venus⁺Foxp3⁻, Venus⁺ Foxp3⁺, and Venus⁻ Foxp3⁺ in the colon significantlydecreased under the GF conditions. Moreover, similar decrease in Venus⁺cells was observed also in the SPF Il10^(venus) mice treated with theantibiotics.

The colonization of Clostridium spp. strongly induced all regulatory Tcell fractions of Venus⁺ Foxp3⁻, Venus⁺ Foxp3⁺, and Venus⁻ Foxp3⁺ in thecolon, and the degrees of the induction thereof were equal to those inthe SPF mice. In addition, it was found that the colonization of thethree strains of Lactobacillus or the colonization of SFB had anextremely small influence on the number of Venus⁺ and/or Foxp3⁺ cells inthe colon. Moreover, the colonization of 16 strains of Bacteroides spp.also induced Venus⁺ cells, but the influence of the colonization wasspecific to Venus⁺ Foxp3⁻ Tr1-like cells. On the other hand, it wasfound that none of the bacterial species tested exerted any significantinfluence on the number of IL-10-producing cells in the small intestinallamina propria (refer to FIG. 26).

Hence, it was shown that the genus Clostridium colonized in the colon ora physiologically active substance derived from the bacteria provided asignal for inducing the accumulation of IL-10⁺ regulatory T cells in thecolonic lamina propria or the expression of IL-10 in T cells. It wasshown that the number of Venus⁺ cells in the small intestine was notsignificantly influenced by the situation where no commensal bacteriawere present or commensal bacteria were decreased, and that IL-10⁺regulatory cells (Tr1-like cells) accumulated in the small intestinallamina propria independently of commensal bacteria.

Example 14: It was investigated whether or not Venus⁺ cells induced bythe genus Clostridium had an immunosuppressive function similar to thatof Venus⁺ cells in the colon of SPF mice. Specifically, CD4⁺ CD25⁻ cells(effector T cells, Teff cells) isolated from the spleen were seeded in aflat-bottomed 96-well plate at 2×10⁴/well, and cultured for three daystogether with 2×10⁴ splenic CD11c⁺ cells (antigen-representing cells)subjected to 30 Gy radiation irradiation treatment, 0.5 μg/ml of ananti-CD3 antibody, and a lot of Treg cells. In addition, for the lastsix hours, the CD4⁺ CD25⁻ cells were cultured, with [³H]-thymidine (1μCi/well) was added thereto. Note that, Treg cells used in Example 14were CD4⁺ GFP⁺ T cells isolated from the spleen of Foxp3^(eGFP) reportermice, or CD4⁺ Venus⁺ T cells in the colonic lamina propria of GFIl10^(venus) mice in which Clostridium spp. were colonized or SPFIl10^(venus) mice. Then, proliferation of the cells was determined basedon the uptake amount of [³H]-thymidine, and represented by a count perminute (cpm) value.

The results show that Venus⁺ CD4⁺ cells of the mice in which the genusClostridium was colonized suppressed in vitro proliferation of CD25⁻CD4⁺ activated T cells. The suppression activity was slightly inferiorto that of GFP⁺ cells isolated from the Foxp3^(eGFP) reporter mice, butequal to that of Venus⁺ cells isolated from the SPF Il10^(venus) mice.Accordingly, it has been shown that the genus Clostridium inducesIL-10-expressing T cells having sufficient immunosuppressive activities,and thereby plays a critical role in maintaining immune homeostasis inthe colon.

Example 15: Next, the influence of the colonization of a large number ofClostridium on the local immune response and the resultant proliferationof Treg cells were investigated.

<Dextran Sulfate Sodium (DSS)-Induced Colitis Model>

First, the DSS-induced colitis model was prepared as described above,and the influence on the model mice of the inoculation of theClostridium and the proliferation of Treg cells was investigated.Specifically, control mice and Clostridium-inoculated mice were treatedwith 2% DSS, then observed and measured for six days for body weightloss, the hardness of stool, and bleeding, and then were evaluatednumerically. In addition, on day 6, the colons were collected,dissected, and analyzed histologically by HE staining.

The results show that the symptoms of the colitis such as body weightloss and rectal bleeding were significantly suppressed in the micehaving a large number of Clostridium (hereinafter also referred to as“Clostridium-abundant mice”) in comparison with the control mice(C57BL/6 mice grown in a conventional environment for six weeks and notinoculated with the fecal suspension). All the features typical forcolonic inflammation, such as shortening of the colon, edema, andhemorrhage, were observed markedly in the control mice in comparisonwith the Clostridium-abundant mice. Moreover, histological features suchas mucosal erosion, edema, cellular infiltration, and crypt loss wereless severe in the DSS-treated Clostridium-abundant mice than in thecontrol mice.

<Oxazolone-Induced Colitis Model>

Next, the oxazolone-induced colitis model was prepared as describedabove, and the influence on the model mice of the inoculation ofClostridium and the proliferation of Treg cells was investigated.Specifically, control mice and Clostridium-inoculated mice weresensitized with oxazolone, and subsequently the inside of the rectumsthereof were treated with a 1% oxazolone/50% ethanol solution. Then,body weight loss was observed and measured. In addition, the colons weredissected, and analyzed histologically by HE staining.

The results show that the colitis proceeded along with persistent bodyweight loss in the control mice. Meanwhile, the body weight loss of theClostridium-abundant mice was reduced. In addition, it was also revealedthat portions having histological diseases such as mucosal erosion,edema, cellular infiltration, and hemorrhage were reduced in the colonof the Clostridium-abundant mice.

Example 16: Next, the influence, on the systemic immune response(systemic IgE production), of the colonization of a large number ofClostridium and the resultant proliferation of Treg cells wasinvestigated. Specifically, as described above, control mice andClostridium-inoculated mice were immunized by administeringalum-absorbed ovalbumin (OVA) twice at a 2-week interval. Then, serawere collected from these mice, and the OVA-specific IgE level thereofwas investigated by ELISA. In addition, splenic cells were collectedfrom the mice in each group, and IL-4 and IL-10 production by in-vitroOVA restimulation was investigated.

Results show that the IgE level was significantly lower in theClostridium-abundant mice than in the control mice. Moreover, the IL-4production by the OVA restimulation was reduced and the IL-10 productionthereby was increased in the splenic cells of the Clostridium-abundantmice sensitized with OVA and alum, in comparison with those of thecontrol mice.

Accordingly, in consideration of the results shown in Example 15 incombination, the induction of Treg cells by Clostridium in the colonplays an important role in local and systemic immune responses.

Example 17: Next, GF Balb/c were colonized with three strains ofClostridium belonging to cluster IV (strains 22, 23 and 32 listed inFIG. 49). Three weeks later, colonic Foxp3⁺ Treg cells were analyzed byFACS. Results show that gnotobiotic mice colonized with three strains ofClostridium showed an intermediate pattern of Treg induction between GFmice and mice inoculated with all 46 strains.

Example 18: Next, it was investigated whether or not a spore-forming(for example, a chloroform resistant) fraction of a fecal sampleobtained from humans had the effect of inducing proliferation oraccumulation of regulatory T cells similar to the spore-forming fractionof the fecal sample obtained from mice.

Human stool from a healthy volunteer (Japanese, male, 29 years old) wassuspended with phosphate-buffered saline (PBS), mixed with chloroform(final concentration 3%), and then incubated in a shaking water bath for60 min. After evaporation of chloroform by bubbling with N₂ gas, thealiquots containing chloroform-resistant (for example, spore-forming)fraction of human intestinal bacteria were orally inoculated intogerm-free (GF) mice (IQI, 8 weeks old). The treated mice were kept in avinyl isolator for 3 weeks. The colon was collected and openedlongitudinally, washed to remove fecal content, and shaken in Hanks'balanced salt solution (HBSS) containing 5 mM EDTA for 20 min at 37° C.After removing epithelial cells and fat tissue, the colon was cut intosmall pieces and incubated with RPMI1640 containing 4% fetal bovineserum, 1 mg/ml collagenase D, 0.5 mg/ml dispase and 40 μg/ml DNase I(all manufactured by Roche Diagnostics) for 1 hour at 37° C. in ashaking water bath. The digested tissue was washed with HBSS containing5 mM EDTA, resuspended in 5 ml of 40% Percoll (manufactured by GEHealthcare) and overlaid on 2.5 ml of 80% Percoll in a 15-ml Falcontube. Percoll gradient separation was performed by centrifugation at 780g for 20 min at 25° C. The interface cells were collected and suspendedin staining buffer containing PBS, 2% FBS, 2 mM EDTA and 0.09% NaN₃ andstained for surface CD4 with Phycoerythrin-labeled anti-CD4 Ab (RM4-5,manufactured by BD Biosciences). Intracellular staining of Foxp3 wasperformed using the Alexa647-labeled anti-Foxp3 Ab (FJK-16s,manufactured by eBioscience) and Foxp3 Staining Buffer Set (manufacturedby eBioscience). The percentage of Foxp3 positive cells within the CD4positive lymphocyte population was analyzed by flow cytometry.

Results show that when the spore-forming (for example, the chloroformresistant) fraction of human intestinal bacteria was colonized in GFmice, the accumulation of Foxp3+ regulatory (Treg) cells in the coloniclamina propria of the mice was induced.

Next, it was investigated what species of bacteria grew by gavaging withchloroform-treated human stool.

Specifically, using a QIAamp DNA Stool mini kit (manufactured byQIAGEN), bacterial genomic DNA was isolated from the human stool from ahealthy volunteer as described above (human stool) or fecal pellets fromGF mice gavaged with chloroform-treated human stool (GF+Chloro.).Quantitative PCR analysis was carried out using a LightCycler 480(manufactured by Roche). Relative quantity was calculated by the ΔCtmethod and normalized to the amount of total bacteria, dilution, andweight of the sample. The following primer sets were used:

total bacteria (SEQ ID NO: 62) 5′-GGTGAATACGTTCCCGG-3′ and(SEQ ID NO: 63) 5′-TACGGCTACCTTGTTACGACTT-3′ Clostridium cluster XIVa(Clostridium coccoides subgroup) (SEQ ID NO: 64)5′-AAATGACGGTACCTGACTAA-3′ and (SEQ ID NO: 65)5′-CTTTGAGTTTCATTCTTGCGAA-3′ Clostridium cluster IV (Clostridium leptum)(SEQ ID NO: 66) 5′-GCACAAGCAGTGGAGT-3′ and (SEQ ID NO: 69)5′-CTTCCTCCGTTTTGTCAA-3′ Bacteroides (SEQ ID NO: 67)5′-GAGAGGAAGGTCCCCCAC-3′ and (SEQ ID NO: 68) 5′-CGCTACTTGGCTGGTTCAG-3′.

Results show that gavaged with chloroform-treated human stool had largeamounts of spore-forming bacteria, such as Clostridium clusters XIVa andIV, and a severe decrease of non-spore-forming bacteria, such asBacteroides, compared with the human stool before chloroform treatment.

Example 19: Human stool (2 g) from a healthy volunteer (Japanese, male,29 y old) was suspended with 20 ml phosphate-buffered saline (PBS),mixed with or without chloroform (final concentration 3%), and incubatedin a shaking water bath for 60 min. The chloroform was then evaporatedby bubbling with N2 gas for 30 min. The suspensions of untreated humanfeces (designated as ‘huUT’) and chloroform-treated human feces(designated as ‘huChloro’) were orally inoculated into Germ-free (GF)mice (IQI, 8 week old) (250 μl/mouse). The suspension of huUT wasinoculated into 4 GF mice, which were numbered from #A1 to #A4, and thatof huChloro was inoculated into 4 GF mice numbered from #B1 to #B4. SuchGF mice which were inoculated with suspensions of feces or the like arealso referred to as “ex-GF mice” hereinafter. Each group of ex-GF micewas separately kept in a vinyl isolator to avoid further microbialcontamination. After 3 weeks, the small intestinal and colonic laminapropria lymphocytes from each mouse were separately collected, andexamined for the expressions of surface CD4 and intracellular Foxp3,Helios, IL-17 and IFN-γ by flow cytometry. For intracellular IL-17 andIFN-γ staining, isolated lymphocytes were stimulated in vitro with PMAand ionomycin for 4 hours. Foxp3 is the transcription factor essentialfor the differentiation and function of Treg cells. Helios is a memberof the Ikaros transcription factor family and Helios-Foxp3+ Treg cellshave been suggested to be Treg cells induced in the periphery [so calledinduced Treg (iTreg) cells]. As shown in FIGS. 1A-D, the percentages ofFoxp3+ Treg cells within CD4+ T cells in the small intestinal andcolonic lamina propria of both groups of ex-GF mice were increased,compared with those in GF mice. Marked increases were also observed forthe percentage of Helios-cells among Foxp3+ Treg cells in smallintestine and colon in both groups of ex-GF mice. Notably, besidesFoxp3+ Treg cells, a significant accumulation of IL-17-expressing CD4+cells (namely, Th17 cells) was observed in exGF+huUT mice, whereas itwas only marginally observed in exGF+huChloro mice (FIGS. 1E, F). Inboth groups of mice, the percentages of IFN-γ+cells in CD4+ cells wereunchanged (FIGS. 1E, G).

Example 20: To investigate whether dead bacteria also have an effect onthe induction of Treg cells, the suspension of chloroform-treated humanfeces was autoclaved (121° C. for 20 min) and orally inoculated into GFmice (once a week for 4 weeks). After 4 weeks, mice were sacrificed, andthe colonic lamina propria lymphocytes from each mouse were examined forthe expression of CD4, Foxp3 and Helios by flow cytometry. As shown inFIG. 2, the inoculation of dead bacteria exhibited no effect on thenumbers of Foxp3+ cells or Helios-Foxp3+ cells. These results do notrule out the possibility that the amount of dead bacteria inoculated wasnot sufficient, but suggest that live bacteria are required for theinduction of Treg cells.

Example 21: To confirm the induction of Treg cells bychloroform-resistant bacteria, another stool was obtained from the sameperson on a different day, treated with chloroform, and inoculated intoIQI GF mice (7 mice, numbered from #C1 to C7). After 3-4 weeks, micefrom #C1 to #C5 were sacrificed, and the small intestinal and coloniclamina propria lymphocytes from each mouse were separately collected,and examined for the expression of CD4 and Foxp3 by flow cytometry.Consistent with the findings in Example 19, colonization withchloroform-treated human feces significantly induced the accumulation ofFoxp3+CD4+ Treg cells in colonic and small intestinal lamina propria(FIG. 3). These results further support the notion thatchloroform-resistant spore-forming bacteria can induce differentiation,proliferation and/or recruitment of Treg cells in intestinal laminapropria.

Example 22: To test whether Treg cell induction by chloroform-resistantspore-forming fraction of human intestinal bacteria is horizontallytransmissible, IQI GF mice (6 mice, numbered from #D1 to #D6) werecohoused for 4 weeks with mice #C6 and #C7 in the same cage in a vinylisolator. Lamina propria lymphocytes from colon and small intestine wereisolated and examined for CD4 and Foxp3. Cohoused mice exhibited asignificant increase in the percentage of Foxp3+ cells among CD4+ cells(FIG. 4). Therefore, Treg cell induction by human intestinal bacteria ishorizontally transmissible. These results let us assume a role ofprominent components of the intestinal microbiota, rather than minorcomponents, for the induction of Treg cells.

Example 23: The frozen stock of cecal content from mouse #C4 was thawed,suspended in 10 times its volume (w/v) of PBS, and passed through a 70μm cell strainer. The suspension was then treated with 3% chloroform,diluted 2000- or 20000-fold with PBS, and orally inoculated into GF IQImice (2.5×10⁵ or 2.5×10⁴ bacterial cells/250 μl/head, respectively). The2000-fold diluted sample was orally inoculated into 4 mice (designatedas exGF+2000, numbered from #E1 to #E4), whereas 20000-fold dilutedsample was inoculated into 8 mice (designated as exGF+20000, numberedfrom #F1 to #F8). After 3 weeks, the intestinal lamina proprialymphocytes were isolated and examined for CD4, Foxp3 and Helios. Both2000- and 20000-fold diluted samples similarly induced a markedaccumulation of Foxp3+CD4+ cells in the intestinal lamina propria (FIG.5). Therefore, the dose of bacteria for oral inoculation can beminimized to less than 2.5×10⁴ bacterial cells.

Example 24: The frozen stock of cecal content from mouse #F3, #F7 or #F8was suspended in 10 times its volume (w/v) of PBS, passed through a 70μm cell strainer, and treated with 3% chloroform. Then, the fecalsuspension from mouse #F3 was orally inoculated into 5 GF mice (numberedfrom #G1 to #G5), that from #F7 mouse into 4 GF mice (numbered from #H1to #H4), and that from #F8 mouse into 4 GF mice (numbered from #I1 to#14). After 4 weeks, lymphocytes from colonic and small intestinallamina propria were isolated and examined for CD4, Foxp3 and Heliosexpression by flow cytometry. All #F, #G, and #H mice exhibited asignificant increase in the percentage of Foxp3⁺ cells among CD4⁺ cellsin the intestinal lamina propria compared with untreated GF mice (FIG.6). Therefore, the Treg cell induction by human intestinal bacteriacolonizing in exGF+20000 mice is also transmissible. Moreover, as shownin the later meta 16S rDNA sequencing data (FIG. 8), these mice commonlyhad bacteria having 16S rDNA sequence similarities with 16S rDNAsequence similarities with 20 species of known bacteria (C. aminophilum,H. saccgarovorans, E. fissicatena, H. filiformis, C. clostridioforme, C.indolis, C. bolteae, Bacteroides sp. MANG, L. bacterium DJF_VP30,Ruminococcus sp. ID8, C. lavalense, C. symbiosum, E. contortum, C.saccharogumia, C. ramosum, F. plautii, C. scindens, Clostridium sp.2335, Clostridium sp. 14616 and cf Clostridium sp. MLG055).

Example 25: A frozen stock of the cecal content from #F8 mouse wasserially diluted with 0.85% NaCl under an aerobic condition and platedonto BL agar. After culture at 37° C. for 2 or 4 days, 50 singlecolonies were observed. Of the 50 colonies, 29 were picked up, culturedfor additional 2 or 4 days at 37° C. by ABCM broth, and stored in EGstock medium (10% DMSO) at −80° C. The genomic DNA from each colony wasisolated, and 16S rRNA coding gene sequence was analyzed. The sequenceof 16S rRNA of each colony revealed that the 29 colonies observed wererepresented by three strains, each having 100% similarity withClostridium ramosum, 99.75% with Clostridium saccharogumia, 100% withFlavonifractor plautii, 99.17% with Clostridium hathewayi, 99.23% withClostridium scindens, or 99.66% with Clostridium sp. 2335. Within the 29colonies that were selected from the original 50 colonies, onlyClostridium saccharogumia, Clostridium ramosum, and Flavonifractorplautii were present (25, 3, and 1 colonies, respectively). These 3isolated strains were propagated, mixed and inoculated into GF IQI mice(4 mice, numbered from #J1 to J4). After 3-4 weeks, the colonic laminapropria lymphocytes were collected, and examined for the expressions ofCD4, Foxp3, and Helios by flow cytometry. Foxp3+ cells or Helios-cellswere not induced or only weakly induced by the colonization of thesestrains of bacteria in the colon (FIG. 7). These results suggest thatthe combination of Clostridium saccharogumia and Clostridium ramosum(both within cluster XVIII) were insufficient to induce Treg cells inthe colon of mice. The effects of Flavonifractor plautii were not clear,since the strain was only represented by 1 of the 29 colonies that wereselected.

Example 26: The frozen glycerol stock of cecal content from #G2 mousewas suspended with PBS, seeded onto BL agar plate, and incubated for 48hours, similarly to the procedure done in Example 19. Different fromExample 19, all bacteria on the plate were collected by scraping with aplate scraper, suspended in TS broth and inoculated into GF IQI mice (4mice, numbering from #K1 to #K4). It should be noted that the bacterialsuspension used in this experiment included bacteria that did notpropagate but survived on the plate. After 4 weeks, lamina proprialymphocytes from colon and small intestine of K1˜K4 mice were isolatedand examined for CD4, Foxp3 and Helios expression. All 4 mice exhibiteda significant increase in the percentages of Foxp3⁺ cells among CD4⁺cells (FIGS. 9A, 9B) and Helios⁻ cells among Foxp3⁺ Treg cells (FIGS.9A, 9C) in the intestinal lamina propria compared with untreated GFmice. Considering that the inoculation of mice with 6 strains ofbacteria propagated on the BL agar plate failed to induce Treg cells,bacteria that did not propagate but survived on the plate might beresponsible for the induction of Treg cells.

Example 27: Bacterial DNA was extracted from the cecal contents of mouse#A1, #C4, #F8, #G2, #H3, #I3, #J3 and #K3. Variable region 1-2 (V1-2) inbacterial 16S rRNA coding gene were amplified by PCR and used astemplate for metasequencing. Resulting sequences (3400 reads for eachsample) were classified into operational taxonomic units (OTUs) on thebasis of sequence similarity (>97% identity). Representative sequencesfrom each OTU were compared with sequences in nucleic acid databasesusing BLAST to determine their closest relatives in known species. Thenumbers of detected reads and the closest relatives for each OTU areshown in Table 1. The relative abundances of OTUs having the sameclosest relative in each cecal sample are shown in FIG. 8. In mouse #A1,153 OTUs (their closest relatives were 93 species) were identified andhalf of them were related to Bacteroides species. In contrast, in mouse#C4, 113 OTUs were identified and most of them were related to speciesbelonging to the family Clostridiaceae. In mouse #F8, #G2, #H3, #I3, #J3and #K3, 97-68 OTUs were identified. In these mice, in which Treg cellaccumulation was observed in the intestine, the majority of bacteriaconsisted of bacteria having 16S rDNA sequence similarities with C.aminophilum, H. saccgarovorans, E. fissicatena, H. filiformis, C.clostridioforme, C. indolis, C. bolteae, Bacteroides sp. MANG, L.bacterium DJF_VP30, Ruminococcus sp. ID8, C. lavalense, C. symbiosum, E.contortum, C. saccharogumia, C. ramosum, F. plautii, C. scindens,Clostridium sp. 2335, Clostridium sp. 14616 and cf Clostridium sp.MLG055.

In mouse #J3, in which Treg accumulation was not observed, 3 OTUs weredetected. Each has the 16S rDNA sequence similarity with C.saccharogumia, C. ramosum or F. plautii. These results suggest that thecombination of these three species are insufficient to induce theintestinal Treg cells accumulation.

Example 28: Bacterial strains were isolated from the cecal contents ofmouse #F8, #G2, #I1 and #K3 using BL agar or EG agar plates. Applicantpicked-up 144 colonies from EG agar plates and 116 colonies from BL agarplates. BLAST search of 16S rRNA coding sequence of these clonesrevealed that they belonged to 17 species, and each had 93-100%similarities with C. indolis, C. bolteae, Bacteroides sp. MANG, L.bacterium DJF_VP30, A. colihominis, Ruminococcus sp. ID8, C. lavalense,C. symbiosum, E. contortum, C. saccharogumia, C. ramosum, F. plautii, C.hathewayi, C. scindens, Clostridium sp. 2335, Clostridium sp. 14616 andcf Clostridium sp. MLG055) (Table 2). They all belonged to Clostridiumclusters IV, XIVa or XVIII (2 species of cluster IV, 12 of cluster XIVa,1 of cluster XVI and 2 of cluster XVIII).

Example 29: Of the colonies selected in Example 28, additional colonieswere picked and isolated and these strains were cultured using EG and BLmedia. BLAST search of 16S rRNA coding sequence of these clones revealedthat they belonged to a total of 31 species (including the speciesmentioned in Example 28), and each had 93-100% similarities withClostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662 (Table 3). The stocks of bacterial strains were storedin 10% glycerol stock plus the media used to grow the cultures, andtubes were stored in a −80° C. freezer.

Example 30: To investigate whether the strains in Example 29 have theability to induce Tregs in GF mice, 31 strains on Table 3 were mixed atequal amounts of media volume using TS media and inoculated into GFmice. A detailed analysis of the 16S rRNA sequences revealed that 8 ofthe 31 strains overlapped with other strains (see Table 3, indicated byan asterisk), resulting in 23 distinct bacterial strains. As shown inFIG. 10, when orally administered to GF mice, the mixture of the 23strains (23 mix) induced very strong levels of Tregs (35-40% in thecolon lamina propria, >10% in the small intestine; FIG. 10). These Tregsobserved with colonization by 23 mix were mostly Helios⁻.

Example 31: To investigate whether the abundant members of theintestinal microbiota in the chloroform-resistant fraction of humanintestinal bacteria, rather than the minor members, drive the inductionof Treg cells, adult GF mice were inoculated with diluted caecal samplesfrom mice that had been inoculated with the chloroform-resistantfraction of human intestinal bacteria (+huChlo mice) as described inexample 19. As shown in FIG. 11, even when the huChlo mice cecal sampleswere diluted (diluted 2×10⁴ and 2×10⁵) to create +2×10⁴ mice and 2×10⁵mice respectively, Tregs were induced in these adult GF mice.

Example 32: To investigate whether the mix of 23 strains in Example 30has the ability to induce Tregs in adult GF IQI mice more effectivelythan Faecalibacterium prausnitzii, a well-known human Clostridia straincharacterized for enhancing regulatory cell functions, 23 strains intable 4 were mixed in equal amounts with media to make a cocktail, whichwas then administered to adult IQI GF mice. For comparison,Faecalibacterium prausnitzii was administered to another group of IQI GFmice. As shown in FIG. 12, when orally administered to adult IQI GFmice, the mixture of the 23 strains (23-mix) induced higher levels ofTregs than Faecalibacterium prausnitzii. Faecalibacterium prausnitzii(+Faecali.) showed negligible levels of Treg induction.

Example 33: To investigate whether the microbiota communities in the+2×104 mice, described in example 31, were stable, serial oralinoculation of adult GF mice was performed to create+2×10⁴-re mice(secondary inoculation) and +2×10⁴-re-re(tertiary inoculation). As shownin FIG. 13 there was significant induction of Tregs in both the+2×10⁴-re mice and the +2×10⁴-re-re mice. To further eliminatenonessential components of the microbiota for Treg cell induction, thecaecal content of +2×10⁴ mice, described in example 31, was againdiluted 2×10⁴-fold and orally inoculated into another set of adult GFmice (+(2×10⁴)² mice). As shown in FIG. 13, the +(2×10⁴)² mice exhibiteda marked accumulation of Treg cells in the colon.

Example 34: To assess the composition of the gut microbiota in +huUT(+hu), +huChlo, +2×10⁴, +2×10⁴-re and +(2×10⁴)², described in example19, example 31, and example 33, bacterial DNA was extracted from thecaecal contents of these adult mice. The variable region (V1-V2) of thebacterial 16S ribosomal DNA (rDNA) was amplified and metasequencingusing a 454 sequencer was performed. The resulting sequences (3400 readsfor each sample) were classified into operational taxonomic units (OTUs)based on sequence similarity (>96% identity). Representative sequencesfrom each OTU were compared with sequences deposited in publiclyavailable 16S and genome databases using BLAST to determine theirclosest species. As shown in FIG. 14, in +hu mice, OTUs belonging toBacteroidetes accounted for about 50% of the caecal microbial community.In contrast, in most OTUs in +huChlo mouse were related to speciesbelonging to Clostridia. In +2×10⁴, +2×10⁴-re and +(2×10⁴)² mice, themajority of bacteria consisted of bacteria having 16S rDNA sequencesimilarities with about 20 species of Clostridia belonging to clusterXIVa (also referred to as C. leptum group), IV, XVI, and XVIII, listedin FIG. 14.

Example 35: A meta analysis of 16S rDNA of caecal contents from miceinoculated with the 23 strains isolated in example 30 (+23-mix mice)confirmed the presence of 17 of the 23 strains listed in FIG. 14 andTable 4. To determine whether these 17 strains could induce Treg cells,a mixture of these 17 strains was inoculated into adult GF mice (+17-mixmice), Each bacterial strain was cultured in 2 mL EG liquid media andgrown to confluence, and then these starter cultures were mixed into a50 mL tube (2 mL×17 strains=34 mL). The bacteria were spun down into apellet and resuspended in 10 mL PBS. A 200 uL aliquot, containing˜1×10⁶-1×10⁷ of each strain, was used to inoculate the adult GF mice. Asshown in FIG. 15, when orally administered to adult IQI, BALB, and B6mice, the mixture of 17 strains was able to induce Tregs in these threemouse models.

Example 36: To investigate whether each of the 17 strains defined inexample 35 could individually induce Tregs, adult GF mice weremonocolonized with one of each of the 17 strains. As shown in FIG. 16,adult GF mice monocolonized with a single strain exhibited low tointermediate levels of Treg. Importantly, no single strain induced Tregsto the same extent as the mix of 17 strains.

Example 37: To investigate whether subsets of the 17 strains describedin example 35 could induce Tregs, randomly selected combinations of 3-5strains were made: 3-mix, 5 mixA, 5-mix B, and 5-mix C, as shown intable 4, and used to inoculate adult GF mice. As shown in FIG. 17, onlythe 5-species mixes induced significant increases in the frequency ofTreg cells, the magnitude of which was intermediate compared with thatobserved in +17-mix mice.

Example 38: To investigate the benefits of administration of the mix ofthe 17 strains described in example 35 (17-mix), adult SPF mice wereorally inoculated with either 17-mix or control media and assessed forthe induction of Foxp3+ Treg cells three weeks later. As shown in FIG.18, there was a significant increase in the frequency of colonic Foxp3⁺Treg (CD4) cells after three weeks of treatment.

Example 39: To evaluate the benefit of administration of 17-mix in ananimal model of allergic diarrhea, adult SPF mice were orally inoculatedwith 17-mix or control media while being treated with ovalbumin (OVA),an inducer of allergic diarrhea. As shown in FIG. 19, the occurrence andseverity of diarrhea (diarrhea score) was significantly reduced in micefed 17-mix relative to control mice.

Example 40: To evaluate the benefit of administration of 17-mix in ananimal model of colitis. Adult SPF mice were orally inoculated witheither 17-mix or control media while being treated with trinitrobenzenesulfonic acid (TNBS), a frequently used experimental inducer of colitis.As shown in FIG. 20, SPF 17-mix mice demonstrated lower mortality thancontrol mice on exposure to TNBS.

Example 41: To evaluate the usefulness of the strains represented in17-mix as a diagnostic and monitoring tool for ulcerative colitis, weexamined the relative abundance of the 17 strains in healthy andulcerative colitis (UC) human subjects using draft genomic sequences ofthe 17 strains and publicly available human faecal microbiome genomesgenerated through the European MetaHIT project. UC subjects (N=20)showed a reduction of the 17 strains compared to healthy subjects(N=15), as shown in FIG. 21.

SEQ ID NOs.: OTU136; OTU46; OTU221; OTU9; OTU296; OTU21; OTU166; OTU73;OTU174; OTU14; OTU55; OTU337; OTU314; OTU195; OTU306; OTU87; OTU86;OTU152; OTU253; OTU259; OTU281; OTU288; OTU334; OTU359; OTU362; orOTU367 are SEQ ID NOs. 19-44, respectively.

INDUSTRIAL APPLICABILITY

As has been described above, the compositions and methods describedherein make it possible to provide an excellent and well-characterizedcomposition for inducing proliferation or accumulation of regulatory Tcells (Treg cells) by utilizing certain human-derived bacteria belongingto the Clostridia class or a physiologically active substance or thelike derived from the bacteria. Since the bacterial composition hasimmunosuppressive effects, the bacterial composition can be used, forexample, to prevent or treat autoimmune diseases or allergic diseases,as well as to suppress immunological rejection in organ transplantationor the like. In addition, healthy individuals can easily and routinelyingest the bacterial composition, such as in food or beverage, (e.g., ahealth food), to improve their immune functions.

TABLE 1A The number of closest relative in known species OTU name Closerelative #C4 #F8 #G2 #H3 #I3 #J3 218 baterium ic1337 9 0 0 0 0 0 104bacterium ic1395 4 0 0 0 0 0 60 64 249 Bacteroides uniformis 0 0 0 0 0 116 Bacteroides vulgattus 0 0 0 0 0 1 233 beta proteobacterium GMD15D04 00 1 0 0 1 138 Bifidibacterium pseudocatenulatum 0 0 0 0 0 2 228 butyrateproducing bacterium M104/1 0 0 1 0 0 3 31 73 cf. Clostridium sp. MLG05510 0 20 0 0 15 227 Clostridiaceae bacterium bSSV31 2 0 0 0 0 0 311Clostridiaceae bacterium FH042 0 0 0 0 0 1 29 52 321 Clostridiaceaebacterium NML 061030 3 0 3 0 0 8 33 156 Clostridiaceae bacterium SH02110 0 0 0 0 0 183 Clostridiaceae bacterium DJF_B152 27 0 0 0 0 0 95 365Clostridium citroniae 2 0 2 0 0 13 106 146 Clostridiumglycyrrhizinilyticum 2 1 0 0 0 0 91 105 102 178 203 292 318 Clostridiuminnocuum 32 0 0 0 0 0 69 80 325 Clostridium lactatifermentans 32 0 0 0 00 47 335 Clostridium methoxybenzovorans 13 0 0 0 0 0 147 175 298 344Clostridium sp. CE6 317 0 0 0 0 1 209 Clostridium sp. CYP2 1 0 0 0 0 0322 Clostridium sp. RT8 5 0 0 0 0 0 223 Clostridium sp. SH-C52 0 1 0 0 00 48 Clostridium xylanovorans 0 0 0 0 0 1 352 Desulfotomaculum sp. CYP11 0 0 0 0 0 132 154 283 Dorea longicatena 0 0 0 0 0 1 164 177Eggerthella lenta 0 0 0 0 0 0 304 Escherichia coli 0 0 0 0 1 0 155Eubacterium dulichum 150 0 0 0 0 0 66 Eubacterium eligens 7 0 0 0 0 0 81219 Eubacterium ramulus 36 0 0 0 0 0 287 Eubacterium siraeum 0 0 0 0 1 053 Eubacterium yurii 0 0 0 1 0 0 41 71 212 222 320 Faecalibacteriumprausnitzii 116 0 0 0 0 0 210 217 271 305 Faecalibacterium sp. DJF_VR20376 0 0 0 0 0 63 197 301 Firmicutes bacterium DJF_VP44 5 0 0 0 0 0 324Fusobacterium periodonticum 0 0 1 0 0 0 180 294 Gram-negative bacteriumcL10-2b-4 0 0 0 0 0 2 190 358 human intestinal bacterium julong 601 8 00 0 0 0 153 184 198 265 Lachnospiraceae bacterium DJF_RP14 2 0 0 0 0 1171 Lactobacillus murinus 0 0 1 0 0 0 17 Odoribacter splanchnicus 13 0 00 0 0 267 Porphyromonas catoniae 0 0 1 0 0 0 145 Prevotellamelaninogenica 0 0 0 0 0 1 8 Prevotella nanceiensis 0 0 1 0 0 0 243Prevotellaceae bacterium DJF_RF17 0 1 0 0 0 0 103 Robinsoniellapeoriensis 1 0 0 0 0 4 98 127 Ruminococcus gnavus 286 0 0 0 0 0 43 99102 159 275 Ruminococcus sp. YE58 1 0 0 0 0 0 341 342 Ruminococcus sp.ZS2-15 53 0 0 0 0 0 252 Streptococcus australis 0 0 1 0 0 0 130 191 272Subdoligranlum sp. DJF_VR33k2 27 0 0 0 0 0 351 Subdoligranlum variabile1 0 0 0 0 0 56 unidentified bacterium ZF3 6 0 0 0 0 0 257 282 284 327329 332 Ruminococcus sp. K-1 322 0 0 0 0 0 87 124 204 259 310 330Eubacterium fissicatena 56 29 86 15 43 28 234 348 Eubacterium contortum2 8 0 6 0 0 90 Lachnospiraceae bacterium A4 2 0 0 0 0 0 2 61 82 92 111163 225 Clostridium aminophilum 565 522 514 380 374 376 266 288 312 336355 359 367 281 296 Clostridium scindens 18 15 29 25 14 17 224 254 264Roseburia hominis 2 1 0 0 1 0 350 Ruminococcus sp. END-1 1 0 1 0 0 0 151242 340 Hydrogenoanaerobacterium saccharovorans 141 205 199 138 175 14044 101 110 119 131 135 Clotridium clostridioforme 12 20 25 75 62 71 137214 260 54 77 97 121 179 187 202 Clotridium symbiosum 31 54 24 19 19 6261 306 326 345 366 27 93 136 182 240 313 Clostridium saccharogumia 257262 200 373 405 307 328 333 Bacteroides capillosus 3 6 3 1 3 0 280Holdemania filliformis 33 46 41 15 31 33 100 120 140 143 166 194 229Clostridium sp. 14616 97 165 493 287 287 153 237 276 297 307 315 319 35467 221 347 Flavonifractor plautii 12 17 34 25 30 29 85 107 butyrateproducing bacterium T2-145 2 0 0 1 0 0 19 40 161 189 195 220 238Clostridium lavalense 75 285 278 475 298 197 262 269 303 334 45 94 109114 125 215 Ruminococcus sp. ID8 30 114 140 135 127 141 248 268 314 337Anaerotruncus colihominis 1 6 3 3 8 2 46 199 213 270 278 Clostridiumramosum 28 74 67 97 110 189 35 37 55 89 129 152 160 Lachospiraceaebacterium 47 268 321 185 232 243 245 279 356 DJF_VP30 12 23 72 86 174201 211 236 Clostridium indolis 13 121 104 253 198 467 246 258 361 364 49 13 14 22 28 38 57 62 76 78 Bacteroides sp. MANG 35 577 530 268 304 226144 186 231 241 362 3 7 15 20 21 24 39 68 70 Clostridium sp. 2335 57 5741 587 637 712 96 113 115 116 123 133 139 162 172 173 206 216 226 247 277286 353 49 117 181 302 339 Clostridium bolteae 0 13 0 30 32 0 74 126 208251 285 Clostridium hathewayi 0 1 0 0 0 0 32 112 Clostridium sp. 14774 00 268 0 0 0 50 165 196 253 Oscillibacter valericigenes 0 14 7 6 7 4 30188 Ruminococcus sp. M-1 0 0 0 0 0 3 Total: 3400 3400 3400 3400 34003400

TABLE 1B The closest relative The number of OTU OTU name in knownspecies Similarity (%) #A1 #C4 #F8 #G2 #H3 #I3 #J3 3 Clostridium sp.2335 98.46 1 0 13 18 10 13 8 9 Bacteroides sp. MANG 98.15 14 0 324 16153 172 159 14 Bacteroides sp. MANG 99.07 4 34 46 401 28 27 14 15Clostridium sp. 2335 96.9 0 0 8 2 0 2 1 21 Clostridium sp. 2335 99.69 1953 325 322 376 410 358 23 Clostridium indolis 97.25 0 0 0 0 3 1 2 38Bacteroides sp. MANG 96.26 0 0 6 0 1 1 4 46 Clostridium ramosum 99.67 4728 70 67 85 101 188 49 Clostridium bolteae 95.98 1 0 7 0 17 28 0 55Lachnospiraceae bacterium 95.53 12 45 120 289 72 85 106 DJF_VP30 57Bacteroides sp. MANG 98.27 3 0 93 0 27 38 20 86 Clostridium indolis98.78 1 0 22 0 43 43 0 87 Eubacterium fissicatena 99.69 1 40 11 39 4 8 089 Lachnospiraceae bacterium 95.18 1 0 4 0 0 2 0 DJF_VP30 92 Clostridiumaminophilum 90.09 0 2 0 1 0 1 0 101 Clostridium clostridioforme 98.76 19 6 3 12 5 12 111 Clostridium aminophilum 91.64 0 1 0 0 0 1 1 114Ruminococcus sp. ID8 95.98 0 4 3 40 0 1 18 119 Clostridiumclostridioforme 98.77 0 1 1 9 0 1 2 125 Ruminococcus sp. ID8 97.25 0 011 12 13 15 43 131 Clostridium clostridioforme 97.23 1 0 1 3 2 1 9 136Clostridium saccharogumia 97.02 10 1 23 16 36 43 12 137 Clostridiumclostridioforme 98.15 1 0 12 10 28 51 47 144 Bacteroides sp. MANG 97.811 0 2 30 1 2 0 152 Lachnospiraceae bacterium 95.55 10 0 129 27 56 135137 DJF_VP30 161 Clostridium lavalense 96.3 0 0 1 1 0 4 0 163Clostridium aminophilum 90.74 0 0 3 0 1 2 0 165 Oscillbactervalaricigenes 90.15 0 9 0 7 0 1 1 166 Clostridium sp. 14616 98.45 2 3514 44 26 32 26 173 Clostridium sp. 2335 98.33 0 0 0 0 0 1 0 174Clostridium indolis 100 0 13 98 103 205 152 465 181 Clostridium bolteae97.56 0 0 5 0 12 2 0 182 Clostridium saccharogumia 94.37 0 2 1 0 3 1 1189 Clostridium lavalense 94.12 0 0 0 0 0 1 0 195 Clostridium lavalense98.47 0 0 47 0 33 31 0 196 Oscillbacter valaricigenes 91.64 1 4 2 0 0 11 199 Clostridium ramosum 98.05 0 0 0 0 5 9 0 202 Clostridium symbiosum97.52 0 0 0 0 0 1 0 204 Eubacterium fissicatena 96.62 0 14 4 30 0 16 15211 Clostridium indolis 94.19 0 0 0 0 1 1 0 214 Clostridiumclostridioforme 95.06 0 1 0 0 0 4 0 221 Flavonifractor plautii 99.69 611 17 34 25 30 29 224 Rosebuna hominis 88.54 0 2 0 0 0 1 0 225Clostridium aminophilum 90.8 0 13 10 8 7 2 1 237 Clostridium sp. 1461699.07 7 0 42 88 100 105 76 246 Clostridium indolis 95.11 0 0 1 1 0 1 0253 Oscillbacter valaricigenes 92.81 9 0 12 0 8 5 2 259 Eubacteriumfissicatena 98.78 1 0 13 17 11 19 13 262 Clostridium lavalense 98.77 016 26 215 25 45 117 268 Ruminococcus sp. ID8 97.82 0 0 36 0 4 100 41 269Clostridium lavalense 97.27 0 0 1 0 2 2 0 277 Clostridium sp. 2335 98.1615 0 146 62 127 125 283 279 Lachnospiraceae bacterium 85.55 1 0 11 0 510 0 DJF_VP30 280 Holdemania fisforms 93.9 14 33 48 41 15 31 33 281Clostridium scindens 99.69 0 11 6 22 15 11 10 286 Clostridium sp. 233597.49 0 0 8 0 3 6 3 287 Eubacterium siraeum 87.3 0 0 0 0 0 1 0 288Clostridium aminophilum 91.33 10 537 394 480 283 249 291 296 Clostridiumscindens 99.69 0 7 9 7 10 3 7 297 Clostridium sp. 14616 94.82 0 21 41 5227 55 22 303 Clostridium lavalense 98.73 2 0 38 0 45 104 54 304Escherichia coli 100 0 0 0 0 0 1 0 306 Clostridium symbiosum 99.38 0 2850 22 1 17 6 307 Clostridium sp. 14616 94.39 1 32 61 62 129 90 25 312Clostridium aminophilum 91.69 0 0 0 0 0 1 0 313 Clostridiumsaccharogumia 98.01 5 254 238 184 127 361 294 314 Ruminococcus sp. ID897.53 0 24 12 88 6 11 39 319 Clostridium sp. 14616 93.19 0 0 1 0 0 5 0326 Clostridium symbiosum 91.67 0 0 0 0 0 1 0 328 Bacteroides capilosus92.9 1 3 4 3 1 2 0 333 Bacteroides capilosus 93.23 0 0 2 0 0 1 0 334Clostridium lavalense 95.37 0 59 50 62 122 111 26 337 Anaerotruncuscolihominis 99.38 2 1 6 3 3 8 2 339 Clostridium bolteae 96.63 0 0 0 0 12 0 340 Hydrogenoanaer bacterium 87 37 141 205 199 138 175 139saccharovorans 353 Clostridium sp. 2335 98.63 7 3 59 87 63 80 54 359Clostridium aminophilum 90.46 1 7 11 18 4 7 8 362 Bacteroides sp. MANG96.14 3 0 100 79 65 64 29 367 Clostridium aminophilum 90.43 2 0 101 3 17111 75

TABLE 2 The Max corresponding similarity Clostridiaceae Origin ofCultured Strain OTU The close relative (%) Cluster mouse sample Mediastrain1 OTU136 Clostridium saccharogumia 99 XVIII #F8 BL strain2 OTU46Clostridium ramosum 100 XVIII #F8, #G2, #J3 BL, EG strain3 OTU221Flavonifractor plautii 100 IV #F8, #G2 BL strain4 OTU9 Clostridiumhathewayi 99 XIVa #F8, #G2 BL strain5 OTU296 Clostridium scindens 99XIVa #F8 BL strain6 OTU21 Clostridium sp. 2335 99 XIVa #F8, #G2 BLstrain7 OTU166 Clostridium sp. 14616 99 XIVa #G2 BL OTU237 strain8 OTU73cf. Clostridium sp. MLG055 99 XVI #G2 BL strain9 OTU174 Clostridiumindolis 99 XIVa #G2, #J3 EG strain10 OTU166 Clostridium sp. 14616 97XIVa #I1 EG OTU181 Clostridium bolteae 98 strain11 OTU14 Bacteroides sp.MANG 99 XIVa #I1 EG strain12 OTU55 Lachnospiraceae bacterium 96 XIVa #I1EG DJF_VP30 strain13 OTU337 Anaerotruncus colihominis 99 IV #I1 EGstrain14 OTU314 Ruminococcus sp. ID8 99 XIVa #I1 EG strain15 OTU195Clostridium lavalense 99 XIVa #I1 EG strain16 OTU306 Clostridiumsymbiosum 99 XIVa #I1 EG strain17 OTU87 Eubacterium contortum 99 XIVa#I1 EG

TABLE 3 Sequenced Similarity to Strain OTU length (bp) Closest StrainSimilarity BLAST other strains Strain1 136 1179 Clostridiumsaccharogumia 99.16 RDPiso Clostridium ramosum JCM1298 96.78 genomeDStrain2 46 1184 Clostridium ramosum 100 RDPiso Clostridium ramosumJCM1298 100 genomeD Strain18 46 492 Clostridium ramosum 100 DDBJ Strain2 Clostridium ramosum 100 genomeDB (>99%) Strain3 211 1182Flavonifractor plautii 100 RDPiso Pseudoflavonifractor capillosus ATCC29799 97.22 genomeD Strain4 9 1154 Clostridium hathewayi 99.31 RDPisoClostridium saccharolyticum WM1 95.06 genomeD Strain11 14 437Bacteroides sp. MANG 99.33 RDPiso Strain 4 Clostridium saccharolyticumWM1 94.9 genomeDB (>99%) Strain19 9 474 Bacteroides sp. MANG 99 DDBJStrain 4 Clostridium saccharolyticum 94.96 genomeDB (>99%) Strain20 14470 Bacteroides sp. MANG 99 DDBJ Strain 4 Clostridium saccharolyticum95.31 genomeDB (>99%) Strain30 362 478 Bacteroides sp. MANG 99 DDBJStrain 4 Clostridium saccharolyticum 94.68 genomeDB (>99%) Strain5 2961182 Clostridium scindens 99.28 RDPiso Lachnospiraceae bacterium5_1_57FAA 99.08 genomeD Strain6 21 1203 Blautia coccoides 99.92 RDPisoLachnospiraceae bacterium 6_1_63FAA 96.43 genomeD Strain7 166 1149Clostridium sp. 14616 99.56 RDPiso Clostridium bolteae ATCC BAA-61399.56 genomeD Strain8 73 1199 cf. Clostridium sp. MLG055 99.42 RDPisoErysipelotrichaceae bacterium 2_2_44A 92.71 genomeD Strain9 174 1189Clostridium indolis 99.24 RDPiso Anaerostipes caccae DSM 14662 97.73genomeD Strain22 86 478 Clostridium indolis 100 DDBJ Strain 9Anaerostipes caccae 96.96 genomeDB (>99%) Strain10 166 491 Clostridiumbolteae 98.03 RDPiso Clostridium bolteae ATCC BAA-613 97.16 genomeDStrain12 96 487 Lachnospiraceae bacterium DJF_VP30 96.08 RDPisoLachnospiraceae bacterium 3_1_57FAA_CT1 99.22 genomeD Strain13 337 490Anaerotruncus colihominis 100 RDPiso Anaerotruncus colihominis DSM 17241100 genomeD Strain14 314 487 Ruminococcus sp. ID3 99.84 RDPisoLachnospiraceae bacterium 2_1_46FAA 96.5 genomeD Strain15 195 488Clostridium lavalense 99.86 RDPiso Clostridium asparagiforme DSM 15981100 genomeD Strain16 306 470 Clostridium symbiosum 99.78 RDPisoClostridium symbiosum WAL-14163 99.56 genomeD Strain17 87 474Eubacterium contortum 99.34 RDPiso Clostridium sp. D5 99.12 genomeDStrain21 87 490 Eubacterium contortum 99 DDBJ strain 17 Clostridium sp.D5 99.13 genomeDB (>99%) Strain23 152 491 Lachnospiraceae bacteriumDJF_VP30 98 DDBJ Lachnospiraceae bacterium 3_1_87FAA_CT1 93.43 genomeDStrain24 253 476 Oscillospiraceae bacterium NML 061048 95 DDBJOscillospiraceae valeriogenes 93.23 genomeD Strain25 259 491 Eubacteriumcontortum 99 DDBJ Clopstridium sp. D5 99.78 genomeD Strain26 281 490Clostridium scindens 97 DDBJ Lachnospiraceae bacterium 5_1_57FAA 98.03genomeD Strain27 288 488 Lachnospiraceae bacterium A4 96 DDBJLachnospiraceae bacterium 5_1_57FAA_CT1 97.45 genomeD Strain28 334 490Clostridium sp. 316002/08 98 DDBJ Clostridiales bacterium 1_7_47FAA99.56 genomeD Strain29 369 488 Lachnospiraceae bacterium A4 93 DDBJLachnospiraceae bacterium 1_1_57FAA_CT1 97.5 genomeD Strain31 367 459Lachnospiraceae bacterium A4 95 DDBJ Strain 39 Lachnospiraceae bacterium3_3_57FAA_CT1 97.8 genomeDB (>99%)

TABLE 4 Sequenced Similarity Corresponding length of 16S Clostridia withthe Database Similarity Mix Strain OUT rDNA (bp) Closest species Clusterclosest species (%) used for BLAST to other strain 23-mix 17-mix 5-mix-A5-mix-B 5-mix-C 3-mix strain1 OTU136 1418 Clostridium saccharogumiaXVIII 99.75 RDPiso X X X X Clostridium ramosum JCM1298 96.78 genomeDBstrain2 OTU46 1184 Clostridium ramosum XVIII 100 RDPiso strain 18 XClostridium ramosum JCM1298 100 genomeDB >99% strain3 OTU221 1427Flavonifractor plautii IV 100 RDPiso X X X X Pseudoflavonifractorcapillosus ATCC 29799 97.22 genomeDB strain4 OTU9 1430 Clostridiumhathewayi XIVa 99.31 RDPiso X X X Clostridium saccharolyticum WM1 95.06genomeDB strain5 OTU296 1433 Clostridium scindens XIVa 99.23 RDPiso XLachnospiraceae bacterium 5_1_57FAA 99.05 genomeDB strain6 OTU21 1428Blautia coccoides XIVa 99.92 RDPiso X X X Lachnospiraceae bacterium6_1_63FAA 96.43 genomeDB strain7 OTU166 1432 Clostridium sp. XIVa 99.56RDPiso X X X Clostridium bolteae ATCC BAA-613 99.56 genomeDB strain8OTU73 1433 cf. Clostridium sp. MLG055 XVI 99.42 RDPiso X X XErysipelotrichaceae bacterium 2_2_44A 92.71 genomeDB strain9 OTU174 1434Clostridium indolis XIVa 99.24 RDPiso X X X Anaerostipes caccae DSM14662 97.73 genomeDB strain10 OTU166 1431 Clostridium bolteae XIVa 98.03RDPiso X Clostridium bolteae ATCC BAA-613 97.15 genomeDB strain11 OTU141430 Bacteroides sp. MANG XIVa 99.33 RDPiso strain 4 Clostridiumsaccharolyticum WM1 94.9 genomeDB >99% strain12 OTU55 1431Lachnospiraceae bacterium DJF_VP30 XIVa 96.08 RDPiso X Lachnospiraceaebacterium 3_1_57FAA_CT1 99.12 genomeDB strain13 OTU337 1418Anaerotruncus colihominis IV 100 RDPiso X X X Anaerotruncus colihominisDSM 17241 100 genomeDB strain14 OTU314 1429 Ruminococcus sp. ID8 XIVa99.54 RDPiso X X X Lachnospiraceae bacterium 2_1_46FAA 96.5 genomeDBstrain15 OTU195 1430 Clostridium lavalense XIVa 99.56 RDPiso X X XClostridium asparagiforme DSM 15981 100 genomeDB strain16 OTU306 1430Clostridium symbiosum XIVa 99.78 RDPiso X X X Clostridium symbiosumWAL-14163 99.56 genomeDB strain17 OTU87 474 Eubacterium contortum XIVa99.34 RDPiso strain 22 Clostridium sp. D5 99.12 genomeDB >99% strain18OTU46 1422 Clostridium ramosum XVIII 100 DDBJ X X Clostridium ramosum100 genomeDB strain19 OTU9 474 Bacteroides sp. MANG XIVa 99 DDBJ strain4 Clostridium saccharolyticum 94.96 genomeDB >99% strain20 OTU14 1430Bacteroides sp. MANG XIVa 99 DDBJ strain 4 Clostridium saccharolyticum95.81 genomeDB >99% strain21 OTU87 490 Eubacterium contortum XIVa 99DDBJ X X Clostridium sp. D5 99.13 genomeDB strain22 OTU86 1424Clostridium indolis XIVa 100 DDBJ strain 9 Anaerostipes caccae 96.96genomeDB >99% strain23 OTU152 1430 Lachnospiraceae bacterium DJF_VP30XIVa 95 DDBJ X Lachnospiraceae bacterium 3_1_57FAA_CT1 98.48 genomeDBstrain24 OTU253 1427 Oscillospiraceae bacterium NML 061048 IV 93 DDBJ XOscillibacter valericigenes 93.23 genomeDB strain25 OTU259 491Eubacterium contortum XIVa 99 DDBJ X Clostridium sp. D5 99.78 genomeDBstrain26 OTU281 1433 Clostridium scindens XIVa 97 DDBJ X X XLachnospiraceae bacterium 5_1_57FAA 98.03 genomeDB strain27 OTU288 1431Lachnospiraceae bacterium A4 XIVa 95 DDBJ X X X Lachnospiraceaebacterium 3_1_57FAA_CT1 97.45 genomeDB strain28 OTU344 1429 Clostridiumsp. 316002/08 XIVa 98 DDBJ X X X Clostridiales bacterium 1_7_47FAA 99.56genomeDB strain29 OTU359 1430 Lachnospiraceae bacterium A4 XIVa 95 DDBJX X X Lachnospiraceae bacterium 3_1_57FAA_CT1 97.8 genomeDB strain30OTU362 1430 Bacteroides sp. MANG XIVa 99 DDBJ strain 4 Clostridiumsaccharolyticum 94.68 genomeDB >99% strain31 OTU367 1430 Lachnospiraceaebacterium A4 XIVa 95 DDBJ strain 29 Lachnospiraceae bacterium3_1_57FAA_CT1 97.8 genomeDB >99%

OTU3 (SEQ ID NO.: 70) GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTAAGACGGATTTC TTCGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGTGAGTAACGCGTGG GTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGGACCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGAT TAGCTAGTTGGAGGGTAACGGCCCACCGAAGGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCAG OTU9 (SEQ ID NO.: 22)GATGAACGCTGGCGGCGGTGCTTAACACATGCAAGTCGAGCGAAGCGGTT TCGAGTGAAGTTTTGGATGGAATTGAAATTGACTTAGCGGCGGACGGGTGAGTAACGCGT GGGTAACCTGCCTTACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGGGCCGCATGGTCTGGTGCGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTG ATTAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCAA OTU14 (SEQ ID NO.: 28)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CAATGAAGTTTTCGGATGGAATTGAAATTGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGGCCGCATGGTCTGGTGTGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAACGGCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGT GACCGGCCACATTGGGGACTGAGACACGGCCCA OTU15 (SEQ ID NO.: 71)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCATTAA GACAGATTTCTTCGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGGCCGCATGGTCTGGTGTGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGACTGAGACACGGCCCAA OTU21 (SEQ ID NO.: 24)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTAA GACAGATTTCTTCGGATTGAAGTCTTTGTGGCTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCA OTU23 (SEQ ID NO.: 72)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCATTTT GGAAGGAAGTTTTCGGATGGAATTCCTTAATGACTGAGTGGCGGACGGGTGAGTAACGCG TGGGGAACCTCCCTACTACAGGGGAGTAACAGCTGGAACGGACTGCTAATACCGCATAAG CGCACAGAATCGCATGATTCGGTGTGAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCTG ATTAGCTGGTTGGCGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAG TGGACGGCCACATTGGGACTGAGACACGGCCCAA OTU38 (SEQ ID NO.: 73)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CAATGAAGTTTTCGGATGGAATTGAAATTGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAG OTU46 (SEQ ID NO.: 20)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGAGCACTT GTGCTCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTAGACAGGGGGATAA CTATTGGAAACGATAGCTAAGACCGCATAGGTACGGACACTGCATGGTGACCGTATTAAA GTGCCTCAAAGCACTGGTAGAGGATGGACTTATGGCGCATTAGCTGGTTGGCGGGGTAAC GGCCCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTG AGACACGGCC CAG OTU49(SEQ ID NO.: 74) GATGAACGCTGGCGGCGTGCCTAACACACGCAAGACGAACGAAGCAATTAAAATGAAGTT TTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGTACCGCATGGTACGTGTGAAAACTACCGGTGGTGTGAGATGGAGTCCCGCGTCT GATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCCACATTGGGGACTGAGACACGGGCCCAA OTU55 (SEQ ID NO.: 29)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTACAC GGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACGGAACCGCATGGTTCCGTGTGAAAAACTACCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACAGCCCA OTU57 (SEQ ID NO.: 75)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CGATGAAGTTTTCGGATGGATTTGAAATCGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGGCCGCATGGTCTGGTGCGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAA OTU73 (SEQ ID NO: 26)GATGAACGCTGGCGGCATGCCTAATACATGCAAGTCGAACGAAGTGAAGA TAGCTTGCTATCGGAGCTTAGTGGCGAACGGGTGAGTAACACGTAGATAACCTGCCTGTA TGACCGGGATAACAGTTGGAAACGACTGCTAATACCGGATAGGCAGAGAGGAGGCATCTC TTCTCTGTTAAAGTTGGGATACAACGCAAACAGATGGATCTGCGGTGCATTAGCTAGTTG GTGAGGTAACGGCCCACCAAGGCGATGATGCATAGCCGGCCTGAGAGGGCGAACGGCCAC ATTGGGACTGAGACACGGCCCAA OTU86 (SEQ ID NO.: 35)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCATTNT TGGAAGGAAGTTTCGGATGGAATTCCTTAATGACTGAGTGGCGGACGGGTGAGTAACGCG TGGGGAACCTACCCTATACAGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAG CGCACAGAATCGCATGATTCGGTGTGAAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCT GATTAGCTGGTTGGCGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGA GTGGACGGCCACATTGGGACTGAGACACGGCCCAA OTU87 (SEQ ID NO.: 34)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTT ACTTAGATTTCTTCGGATTGAAAGTTTTGCGACTGAGCGGCGGACGGGTGAGTAACGCGT GGGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGA CCACAGTACCGCATGGTACAGTGGGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTG ATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU89 (SEQ ID NO.: 76)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCATTTT GGAAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTG GGAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAACACCGCATAAGCG CACGGAACCGCATGGTTCTGTGTGAAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTG ATTAGCCAGTTGGCGAGGGTAACGGCCTACCAAAGACGACGATCAGTAGCCGGCCTGAGA GGGTGAACGGCCACATTGGGACTGAGACACGGCCCAA OTU92 (SEQ ID NO.: 77)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGTTATGC AGAGGAAGTTTTCGGATGGAATCGGCGTAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAGGCAGTGTGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGAT TAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGT GACCGGCCACATTGGGACTGAGACACGGCCCA OTU101 (SEQ ID NO.: 78)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AGATGAAGTTTTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTGCCGCATGGCAGTGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGGCGGGGTAACGGCCACCGAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACACTGGGACTGAGACACGGCCCA OTU111 (SEQ ID NO.: 79)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTACAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGCTGGTTGGCGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAGT GGACGGCCACATTGGGACTGAGACACGGCCCA OTU114 (SEQ ID NO.: 80)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGT TTTCAGAATCTTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCA OTU119 (SEQ ID NO.: 81)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AGATGAAGTTTTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTGCCGCATGGCAGTGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGCGGGGTAACGGCCCGACCAAAGCGACGGATCAGTAGCCGACCTGAGAGG GTNACCGGCCACATTGGGACTGAGACACGGCCCA OTU125 (SEQ ID NO.: 82)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGT TTTCAGAATCTTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGTAAAGGCTACCGAAGCCGACGATCAGTAGCCGACCTGACGAGGG TGACCGGCCACGATTGGGACTGAGACACGGCCCAA OTU131 (SEQ ID NO.: 83)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AGATGAAGTTTTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTGCCGCATGGCAGTGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGCGGGTAACGGCCACCGAAAGCGACGATCAGTAGCCGACCTGACGAGGGT NACCGGCACATTGGGACTGAGACACGGCCCAA OTU136 (SEQ ID NO.: 19)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGAGCACTT GTGCTCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTTTACAGGGGGATAA CTATTGGAAACGATAGCTAAGACCGCATAGGTAAAGATACCGCATGGTAAGTTTATTAAA AGTGCCAAGGCACTGGTAGAGGATGGACTTATGGCGCATTAGCTAGTTGGTGAGGTAACG GCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGA GACACGGCCC AG OTU137(SEQ ID NO.: 84) GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTAAGATGAAGTT TTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGTGCCGCATGGCAGTGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTG ATTAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCAA OTU144 (SEQ ID NO.: 85)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CGATGAAGTTTTTGGATGGAATTGAAATTGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGGCCGCATGGTCTGGTGCGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAACGGCCCACCGAAGCCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCACATTGGGACCTGAGACACGGGCCCA OTU152 (SEQ ID NO.: 36)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTAGAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACGGAACCGCATGGGTTCTGTGTGAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCAA OTU161 (SEQ ID NO.: 86)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAA OTU163 (SEQ ID NO.: 87)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTACAC GGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACGGAACCGCATGGTTCCGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGT GACCGGCCACATTGGGACTGAGACACGGCCCA OTU165 (SEQ ID NO.: 88)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAGCACCCT TGACTGAGGTTTCGGCCAAATGATAGGAATGCTTAGTGGCGGACTGGTGAGTAACGCGTG AGGAACCTACCTTCCAGAGGGGACGAACAGTTGGAACGACTGCTAATACCGCATGACGCA TGACCGGGGCGATCCCGGGCCGATGTCAAAGATTTTATTCGCTGGAAGATGGCCTCGCGT CTGATTAGCTAGATGGTGGGGTAACGGCCCACCATGGCGACGATCAGTAGCCGGACTGAG AGGTTGACCGGCCACATTGGGACTGAGATACGGCCCA OTU166 (SEQ ID NO.: 25)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AAATGAAGTTTCGGATGGATTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGA TAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCA CAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGATT AGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGGTG ACCGGCCACGATTGGGACTGAGACACGGCCCA OTU173 (SEQ ID NO.: 123)GACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGAGTTGTGT TGAAAGCTTGCTGGATATACAACTTAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTG CCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGGATC GCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAACTAGT TGGAGGGGTAACGGCCCACCAAGGCGACGAGTCAGTAGCCGGCCTGAGAGGGTGAACGGC CACGATTGGGACTGAGACACGGCCCAG OTU174 (SEQ ID NO.: 27)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCATTTT GGAAGGAAGTTTTCGGATGGAATTCCTTAATGACTGAGTGGCGGACGGGTGAGTAACGCG TGGGGAACCTGCCCTATACAGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAG CGCACAGAATCGCATGATTCGGTGTGAAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCT GATTAGCTGGTTGGCGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAG AGTGGACGGCCACATTGGGACTGAGACACGGCCCA OTU181 (SEQ ID NO.: 89)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTT AAAATGAAGTTTTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGT GGATAACCTGCCTCACGACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAG CGCACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCT GATTAGCCAGTTGCGGGGTAACGGCCCACCGAAAGCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCCACATTGGGGACTGAGACACGGCCCAA OTU182 (SEQ ID NO.: 90)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGGGCAGCA ATGCCCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTTTACAGGGGGATAA CTATTGGAAACGATAGCTAAGACCGCATAGGTAAAGATACCGCATGGTAAGTTTATTAAA AGTGCCAAGGCACTACGAGGGAGTAGTGATATGCGCATAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGA CACGGCCCAG OTU189(SEQ ID NO.: 91) GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTTAGATGAAGTT TTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACACAGTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCAG OTU195 (SEQ ID NO.: 32)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACGAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTG ATTAGCCAGTTGGCGGGTAACGGCCCACCGAAAGCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCAA OTU196 (SEQ ID NO.: 92)GACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAGCACCCC TGAATGAGGTTTCGGCCAAAGGAAGGGAATGCTTAGTGGCGGACTGGTGAGTAACGCGTG AGGAACCTGCCTTTCAGAGGGGACAACAGTTGGAAACGACTGCTAATACCGCATGACACA TGAATGGGGCATCCCATTGATGTCAAAGATTTATCGCTGAAAGATGGCCTCGCGTCCCAT TAGCTAGTAGGCGGGGTAACGGCCCACCTAGGCGACGATGGGTAGCCGGACTGAGAGGTT GACCGGCCACATTGGGACTGAGATACGGCCCA OTU199 (SEQ ID NO.: 93)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGAGCACTT GTGCTCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTAGACAGGGGGAGTA ACTATTGGAACGATAGCTAAGACCGCATAGGTACGGACACTGCGTGGTGACCGTATTAAA AGTAGCCTCAAAGACACTGGTAGAGGATGGACTTATGGCGCATTAGCTGGTTGGCGGGGT AACGGCCCACCCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGG ACTGAGACAC GGCCCAGOTU202 (SEQ ID NO.: 94)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTT AACGGAAGTTTTCGGATGGAAGTTGAATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTGTACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTACAAGATGGACCCGCGTCTGA TTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAG OTU204 (SEQ ID NO.: 95)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTAA GACGGATTTCTTCGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGAC CACAGTACCGCATGGTACAGTGGGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGACTGAGACACGGCCCA OTU211 (SEQ ID NO.: 96)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CGATGAAGTTTTCGGATGGATTTGAAATCGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAGCG CACAGAATCGCATGATTCGGTGCGAAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCTGA TTAGCTGGTTGGCGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAG TGGACGGCCACATTGGGACTGAGACACGGCCCAA OTU214 (SEQ ID NO.: 97)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AGATGAAGTTTTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGAGTAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGGGCTGCATGGCCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTG ATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGG GTGAACGGCCACATTGGGACTGAGACACGGCCCA OTU221 (SEQ ID NO.: 21)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGGTGCTCA TGACGGAGGATTCGTCCAACGGATTGAGTTACCCAGTGGCGGACGGGTGAGTAACGCGTG AGGAACCTGCCTTGGAGAGGGGAATAACACTCCGAAAGGAGTGCTAATACCGCATGATGC AGTTGGGTCGCATGGCTCTGACTGCCAAAGATTTATCGCTCTGAGATGGCCTCGCGTCTG ATTAGCTAGTAGGCGGGGTAACGGCCCACCTAGGCGACGATCAGTAGCCGGACTGAGAGG TTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU224 (SEQ ID NO.: 98)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACCTT GGCGGATTTCTTCGGATTGAAGCCTTGGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCG CACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGCGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGACAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCA OTU225 (SEQ ID NO.: 99)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAAGTTATG CAGAGGAAGTTTTCGGTATGGAATCGGCGTAACTTAGTGGCGGACGGGTGAGTAACGCGT GGGAAACCTGCCCTGTACCGGGGGAGTAACACTTAGAATAGGTGCTAATACCGCATAAGC GCACAGCTTCACATGAGGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTG ATTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGG GTGAACGGCCACATTGGGACTGAGACACGGCCCA OTU237 (SEQ ID NO.: 100)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTG AAGGAAGTTTTCGGATGGAATTCGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGA TAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCA CAGTGCCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGATT AGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGGTG ACCGGCCACATTGGGACTGAGACACGGCCCAA OTU246 (SEQ ID NO.: 101)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGTTATGC AGAGGAAGTTTTCGGATGGAATCGGCGTAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTATACAGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAGCGC ACAGAATCGCATGATTCGGTGTGAAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCTGAT TAGCTGGTTGGCGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAGT GGACGGCCACATTGGGACTGAGACACGGCCCAA OTU253 (SEQ ID NO.: 37)GACGAACGCTGGCGGCGTGCTTAACACATGCAAATCGAACGGAGCACCCT TGACTGAGGTTTCGGCCAAATGATAGGAATGCTTAGTGGCGGACTGGTGAGTAACGCGTG AGGAACCTGCCTTCCAGAGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATGACGC ATGACCGGGGCATCCCGGGCATGTCAAAGATTTTATCGCTGGAAGATGGCCTCGCGTCTG ATTAGCTAGATGGTGGGGTAACGGCCCACCATGGCGACGATCAGTAGCCGGACTGAGAGG TTGACCGGCCACATTGGGACTGAGATACGGGCCCAG OTU259 (SEQ ID NO.: 38)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTT ACTTAGATTTCTTCGGATTGAAAAGTTTTGCGACTGAGCGGCGGACGGGTGAGTAACGCG TGGGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAG ACCACGGTACCGCATGGTACAGTGGGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCT GATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCACATTGGGACCTGAGACACGGCCCAA OTU262 (SEQ ID NO.: 102)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTACCGCATGGTACAGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGGACCTGAGACACGGCCCA OTU268 (SEQ ID NO.: 103)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGT TTTCAGAATCTTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAAAGGCCTACCAAGCCGACGATCAGTAGCCGACCTGAGACGG GTGACCGGCACATTGGGGACTGAGACACGGGCCCAA OTU269 (SEQ ID NO.: 104)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACGAACAGTTAGAAATAGACTGCTAATACCGCATAAG CGCACAGTACCGCATGGTACAGTGTGAAAAACTACCGGTGGTGTGAGATGGATCCGCGCT GATTAGTCCAGTTGGCGGGGTAACGGCCGACCAAAGCGACGATCAGTAGCCGACCTGAGA GGGTGACCGGCCGACAGTTGGGACTGAGACACGGCCCAA OTU277 (SEQ ID NO.: 105)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTAA GACGGATTTCTTTGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGATCGCATGGTCTGGTGTGGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTG ATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGG GTGAACGGCCACGATTGGGACTGAGACACGGCCCAG OTU279 (SEQ ID NO.: 106)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTAGAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAACCTGCCCTGTACCGGGGGAGTAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACGGAACCGCATGGTTCTGTGTGAAAAACTACCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCA OTU280 (SEQ ID NO.: 107)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTTGTAAA GGAGCTTGCTTCTTTACGAGGAGTGGCGAACGGGTGAGTAATACATAAGCAATCTGCCCA TCGGCCTGGGATAACAGTTGGAAACGACTGCTAATACCGGATAGGTTAGTTTCTGGCATC AGGGACTAATTAAAGTTGGGATACAACACGGATGGATGAGCTTATGGCGTATTAGCTAGT AGGTGAGGTAACGGCCCACCTAGGCGATGATACGTAGCCGACCTGAGAGGGTGACCGGCC ACATTGGGACTGAGACACGGCCCAA OTU281 (SEQ ID NO.: 39)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCGCTTC CGCCTGATTTTCTTCGGAGATGAAGGCGGCTGCGACTGAGTGGCGGACGGGTGAGTAACG CGTGGGCAACCTGCCTTGCACTGGGGGATAACAGCCAGAAATGGCTGCTAATACCGCATA AGACCGAAGCGCCGCATGGCGCTGCGGCCAAAGCCCCGGCGGTGCAAGATGGGCCCGCGT CTGATTAGGTAGTTGGCGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAG AGGGTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU286 (SEQ ID NO.: 108)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTAA GACGGATTTCTTCGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGATCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGGCCCAA OTU287 (SEQ ID NO.: 109)GACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGACACATCC GACGGAATAGCTTGCTAGGAAGATGGATGTTGTTAGTGGCGGACGGGTGAGTAACACGTG AGCAACCTGCCTCGGAGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATACGGT GGTCGGGGGACATCCCCTGGCCAAGAAAGGATTATATCCGCTCTGAGATGGGCTCGCGTC TGATTAGCTAGTTGGCGGGTAATGGCCCGACCGAAGGCAACGATCAGTAGCCGGACTGAG AGGTTGAACGGCCACATTGGGACTGAGACACGGCCCCAG OTU288 (SEQ ID NO.: 40)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGTTATGC AGAGGAAGTTTTCGGATGGAATCGGCGTAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCA OTU296 (SEQ ID NO.: 23)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCGCCTG GCCCCGACTTCTTCGGAACGAGGAGCCTTGCGACTGAGTGGCGGACGGGTGAGTAACGCG TGGGCAACCTGCCTTGCACTGGGGGATAACAGCCAGAAATGGCTGCTAATACCGCATAAG ACCGAAGCGCCGCATGGCGCAGCGGCCAAAGCCCCGGCGGTGCAAGATGGGCCCGCGTCT GATTAGGTAGTTGGCGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU297 (SEQ ID NO.: 110)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATCTT ATAGGAAGTTTTCGGATGGAATATGGGATGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGAGTAACAGTTAGAAATGGCTGCTAATACCCCACTAAG CGCACGGTACCGCATGGTACGGTGTGAAAAACCCAGGTGGTGTGAGATGGATCCGCGTCT GATTAGCCAGTTGGCGGGGTAACGGCCCGACCAAACGCGACGATCAGTAGCCGACCTGAG AGGGTGACCGGCCGACATTGGGACTGAGACACGGCCCA OTU303 (SEQ ID NO.: 111)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTACCGCATGGTACAGCGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCACATTGGGGACTGAGACCACGGGCCCAA OTU304 (SEQ ID NO.: 112)ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGA AGCAGCTTGCTGCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCC GATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAA GAGGGGGACCTTAGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTG GGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACT GGAACTGAGA CACGGTCCAGOTU306 (SEQ ID NO.: 33)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCGACTT AACGGAAGTTTTCGGATGGAAGTTGAATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTGTACTGGGGGACGAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTACAAGATGGACCCGCGTCTG ATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU307 (SEQ ID NO.: 113)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATCTT ATAGGAAGTTTTCGGATGGAATATGGGATGACTGAGTGGCGGACGGGTGAGTAACGCGTG GAGTAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGGCTGCTAATACCCCATAAGC GCACAGTACCGCATGGTACGGTGTGAAAAACCCAGGTGGTGTGAGATGGATCCGCGTCTG ATTAGCCAGTTGGCGGGTAACGGCCGACCAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCACGATTGGGACCTGAGACACGGGCCCA OTU312 (SEQ ID NO.: 114)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTATAT CGAGGAAGTTTTCGGATGGAATCAGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGAT TAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCAG OTU313 (SEQ ID NO.: 115)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGGGCAGCA ATGCCCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTTTACAGGGGGATAA CTATTGGAAACGATAGCTAAGACCGCATAGGTAAAGATACCGCATGGTAAGTTTATTAAA GTGCCAAGGCACTGGTAGAGGATGGACTTATGGCGCATTAGCTAGTTGGTGAGGTAACGG CTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAG ACACGGCCCA A OTU314(SEQ ID NO.: 31) GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGTTTTCAGAATC TTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAAGGCCGTACCAAGCCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGGACTGAGACACGGCCCA OTU319 (SEQ ID NO.: 116)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTAGAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGTACCGCATGGTACAGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGAT TAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGGT GACCGGCACATTGGGACTGAGACACGGCCCAA OTU326 (SEQ ID NO.: 117)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AAATGAAGTTTTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAA OTU328 (SEQ ID NO.: 118)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAGTGCCTT AGAAAGAGGATTCGTCCAATTGATAAGGTTACTTAGTGGCGGACGGGTGAGTAACGCGTG AGGAACCTGCCTCGGAGTGGGGAATAACAGACCGAAAGGTCTGCTAATACCGCATGATGC AGTTGGACCGCATGGTCCTGACTGCCAAAGATTTATCGCTCTGAGATGGCCTCGCGTCTG ATTAGCTTGTTGGCGGGGTAATGGCCCACCAAGGCGACGATCAGTAGCCGGACTGAGAGG TTGGCCGGCCACATTGGGACTGAGACACGGCCCA OTU333 (SEQ ID NO.: 119)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAGTGCTCA TGACAGAGGATTCGTCCAATGGAGTGAGTTACTTAGTGGCGGACGGGTGAGTAACGCGTG AGTAACCTGCCTTGGAGTGGGGAATAACAGGTGGAAACATCTGCTAATACCGCATGATGC AGTTGGGTCGCATGGCTCTGACTGCCAAAGATTTATCGCTCTGAGATGGACTCGCGTCTG ATTAGCTGGTTGGCGGGTAACGGCCACCAAGGCGACGATCAGTAGCCGGACTGAGAGGTT GGCCGGCCACATTGGGACTGAGACACGGCCCAG OTU334 (SEQ ID NO.: 41)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATCCC ATAGGAAGTTTTCGGATGGAATATGGGATGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGGCTGCTAATACCGCATAAGCG CACAGTACCGCATGGTACGGTGTGAAAAACCCAGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGGACTGAGACACGGCCCA OTU337 (SEQ ID NO.: 30)GACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGAGCTTACG TTTTGAAGTTTTCGGATGGATGAATGTAAGCTTAGTGGCGGACGGGTGAGTAACACGTGA GCAACCTGCCTTTCAGAGGGGGATAACAGCCGGAAACGGCTGCTAATACCGCATGATGTT GCGGGGGCACATGCCCCTGCAACCAAAGGAGCAATCCGCTGAAAGATGGGCTCGCGTCCG ATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCGGTAGCCGGACTGAGAGG TTGAACGGCCACATTGGGACTGAGACACGGCCCAG OTU339 (SEQ ID NO.: 120)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AAATGAAGTTTTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGAT TAGCCAGTTGCGGGGTAACGGCCCGACCAAGCGACGATCAGTAGCCGACCGTGAGAGGTG ACCGGCCCACATTGGGACTGAGACACGGCCCAA OTU340 (SEQ ID NO.: 121)GACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGAGTTGTGT TGAAAGCTTGCTGGATATACAACTTAGTGGCGGACGGGTGAGTAACACGTGAGTAACCTG CCTCTCAGAGTGGAATAACGTTTGGAAACGAACGCTAATACCGCATAACGTGAGAAGAGG GCATCCTCTTTTTACCAAAGATTTATCGCTGAGAGATGGGCTCGCGGCCGATTAGGTAGT TGGTGAGATAACAGCCCACCAAGCCGACGATCGGTAGCCGGACTGAGAGGTTGATCGGCC ACATTGGGACTGAGACACGGCCCAG OTU353 (SEQ ID NO.: 122)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACCTT GACGGATTCTTCGGATTGAAGCCTTGGTGACTGAGCGGCGGACGGGTGAGTAACGCGTGG GTAACCTGCCTCATACAGGGGGGATAAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGGACCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTG ATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGG GTGAACGGCCACATTGGGACTGAGGACACGGCCCA OTU359 (SEQ ID NO.: 42)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTATAT CGAGGAAGTTTTCGGATGGAATCAGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGGAGAGG GTGAACGGCCACATTGGGACTGAGACACGGCCCG OTU362 (SEQ ID NO.: 43)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CGATGAAGTTTTCGGATGGATTTGAAATCGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGGGCCGCATGGTCCGGTGTGAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGATT AGGTAGTTGGTGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGTGA CCGGCCACATTGGGACTGAGACACGGCCCAA OTU367 (SEQ ID NO.: 44)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTACAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCCGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAACTCCGGTAGGTACAGGATGGTCCCGCGTCTGAT TAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGGT CAACGGCCACATTGGGACTGAGACACGGCCCAA

What is claimed is:
 1. A pharmaceutical composition comprising apurified bacterial mixture consisting of bacteria comprising 16S rDNAsequences of at least 95% homology to SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42. 2.The pharmaceutical composition of claim 1, wherein the bacteria arehuman-derived bacteria.
 3. The pharmaceutical composition of claim 1,wherein the bacteria are isolated from a chloroform-treated fecalsample.
 4. The pharmaceutical composition of claim 1, wherein thebacteria are isolated from a heat-treated fecal sample.
 5. Thepharmaceutical composition of claim 1, wherein at least a portion of thebacteria are in spore-form.
 6. The pharmaceutical composition of claim1, further comprising a pharmaceutically acceptable excipient.
 7. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is formulated for oral administration.